Library of ph responsive polymers and nanoprobes thereof

ABSTRACT

The present disclosure relates to polymers which contain a hydrophobic and hydrophilic segment which is sensitive to pH. In some aspects, the polymers form a micelle which is sensitive to pH and results in a change in fluorescence based upon the particular pH. In some aspects, the disclosure also provides methods of using the polymers for the imaging of cellular or extracellular environment or delivering a drug.

This application is a continuation of U.S. application Ser. No.15/369,701, filed Dec. 5, 2016, which is a continuation of InternationalApplication No. PCT/US2015/034575, filed Jun. 5, 2015, which claims thebenefit of priority from U.S. Provisional Application Ser. No.62/009,019, filed on Jun. 6, 2014, the entire contents of each of whichare incorporated herein by reference.

This invention was made with government support under Grant Number R01EB013149 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND 1. Field

The present disclosure relates generally to the fields of molecular andcellular biology, cancer imaging, nanotechnology, and fluorescencesensors. More particularly, it relates to nanoplatforms for thedetection of pH changes.

2. Description of Related Art

Fluorescence imaging has become an important tool in the study ofbiological molecules, pathways and processes in living cells thanks toits ability to provide spatial-temporal information at microscopic,mesoscopic and macroscopic levels (see, e.g., Tsien, R. Y. Nat. Rev.Mol. Cell Biol. 2003, 4, SS16; Weissleder, R., Nature 2008, 452, 580;Fernandez-Suarez, M., Nat. Rev. Mol. Cell Biol. 2008, 9, 929). Recently,activatable imaging probes that are responsive to physiological stimulisuch as ionic and redox potentials, enzymatic expressions, and pH havereceived considerable attention to probe cell physiological processes(see, e.g., de Silva, A. P., Chem. Rev. 1997, 97, 1515; Zhang, J., Nat.Rev. Mol. Cell Biol. 2002, 3, 906; Lee, S., Chem. Commun. 2008, 4250;Kobayashi, H.; Chem. Res. 2010, 44, 83; Lovell, J. F., Chem. Rev. 2010,110, 2839; Ueno, T., Nat. Methods 2011, 8, 642). Among these stimuli, pHstands out as an important physiological parameter that plays a criticalrole in both the intracellular (pH_(i)) and extracellular (pH_(e))milieu (Alberts, B., Molecular Biology of the Cell; 5th ed.; GarlandScience: New York, 2008).

Although various pH-sensitive fluorescent probes have been reported(Kobayashi, H., Chem. Rev. 2010, 110, 2620; Han, J. Y., Chem. Rev. 2010,110, 2709), their pH sensitivity primarily arises from ionizableresidues with pH-dependent photo-induced electron transfer (PeT)properties to the fluorophores. One potential drawback for thesefluorescent agents is their broad pH response (ΔpH˜2) as dictated by theHenderson-Hasselbalch equation (Atkins, P., Physical Chemistry; OxfordUniversity Press, 2009). This lack of sharp pH response makes itdifficult to detect subtle pH differences between the acidicintracellular organelles (e.g., <1 pH difference between early endosomesand lysosomes) (Maxfield, F. R., Nat. Rev. Mol. Cell Biol. 2004, 5, 121;Casey, J. R., Nat. Rev. Mol. Cell Biol. 2010, 11, 50) or pHe in solidtumors (6.5-6.9) (Webb, B. A., Nat. Rev. Cancer 2011, 11, 671; Zhang,X., J. Nucl. Med. 2010, 51, 1167) over normal tissue environment (7.4).Moreover, simultaneous control of pH transition point and emissionwavelengths (in particular, in the near IR range) is difficult for smallmolecular dyes. Recent attempts to develop pH-sensitive fluorescentnanoparticles primarily employ polymers conjugated with small molecularpH-sensitive dyes (Srikun, D., J. Chem. Sci. 2011, 2, 1156; Benjaminsen,R. V., ACS Nano 2011, 5, 5864; Albertazzi, L., J. Am. Chem. Soc. 2010,132, 18158; Urano, Y., Nat. Med. 2009, 15, 104) or the use ofpH-sensitive linkers to conjugate pH-insensitive dyes (Li, C., Adv.Funct. Mater. 2010, 20, 2222; Almutairi, A., J. Am. Chem. Soc. 2007,130, 444.). These nanoprobe designs also yield broad pH response andlack the ability to fine-tune pH transition point.

Recently, the use of polymers to create a pH responsive system has beendescribed in WO 2013/152059, which produces a relatively narrow range ofpH transition points based upon the specific monomer used but lacks theflexibility to fine-tune the pH transition point specifically.

Furthermore, imaging of tumor cells can provide enhanced methods ofdelineating the tumor boundaries and increasing the efficacy of surgeryto resect a tumor. A variety of methods have been proposed to assist inthe delineation of tumor boundaries. Conventional imaging modalitiessuch as CT, MRI or ultrasound using image navigators such as theBrainlab™ first use pre-operative images followed by the intra-operativeuse of surgical fiducial markers to guide resection of skull base andsinus cancers as well as brain tumors. A major drawback is that onlytumors that are immobile relative to firm bony landmarks can beaccurately imaged and the pre-operative images cannot be updated toaccount for intra-operative manipulations to provide real-time feedback.Intra-operative MRI is being used in a few centers for imaging braintumors but requires expensive installation of magnets into the operativesuite for real time imaging and a recent review suggest that this may beof marginal benefit over conventional surgical navigation (Kubben etal., 2011). Ultrasound has been used to assess tumor depth for oralcavity HNSCC but is difficult to use in less accessible primary sites ofthe head and neck (Lodder et al., 2011).

These anatomy-based imaging modalities have great resolution but providelittle disease specific information. Optical imaging strategies haverapidly been used to image tissues intra-operatively based on cellularimaging, native autofluorescence, and Raman scattering (Vahrmeijer etal., 2013; Nguyen & Tsien, 2013; Dacosta et al., 2006; Draga et al.,2010; Haka et al., 2006; Schwarz et al., 2009 and Mo et al., 2009).Unfortunately, using tissue autofluorescence for tumor margin detectionis limited by high false positive and false negative results due to thelack of robust spectroscopic differences between cancer and normaltissues (Liu et al., 2010; Kanter et al., 2009; Ramanujam et al., 1996and Schomacker et al., 1992).

A variety of exogenous fluorophores have been developed forintra-operative margin assessment. Most common strategies have focusedon cell-surface receptors such as folate receptor-α (FR-α) (van Dam etal., 2011), chlorotoxin (Veiseh et al., 2007), epidermal growth factorreceptor (EGFR) (Ke et al., 2003 and Urano et al., 2009), Her2/neu(Koyama et al., 2007), tumor associated antigens (e.g.,prostate-specific membrane antigen, PSMA) (Tran Cao et al., 2012,carcinoembryonic antigen and carbohydrate antigen 19-9 (CA19-9) (TranCao et al., 2012; McElroy et al., 2008). Among these, folate-FITC andchlorotoxin-Cy5.5 conjugate have already advanced to Phase I clinicaltrials in surgery of ovarian and skin cancers, respectively. Despitethese successes, one of the major limitations is the lack of broad tumorapplicability in cancer patients. For the cell-surface receptor strategyof tumor visualization, lack of a uniform marker makes it difficult tocreate a universal platform to visualize tumors with a diverseoncogenotypes and anatomical sites. In addition, the relatively low(fmol-nmol) and highly variable expression levels (100-300 folds) makesit challenging for conventional stoichiometric strategy (e.g., 1:1 forligand:receptor) without signal amplification. This is particularlychallenging for mAb-dye conjugates (e.g., Erbitux-ICG) due to the longcirculation times of humanized mAb that raise the blood backgroundbecause of the always-on probe design.

As such, new polymers that can generate pH responsive systems for theimaging of tumors are of value to development diagnostic and imagingprotocols.

SUMMARY

In some aspects, the present disclosure provides a polymer of theformula:

wherein: R₁ is hydrogen, alkyl_((C≤12)), cycloalkyl_((C≤12)),substituted alkyl_((C≤12)), substituted cycloalkyl_((C≤12)), or

or a metal chelating group; n is an integer from 1 to 500; R₂ and R₂′are each independently selected from hydrogen, alkyl_((C≤12)),cycloalkyl_((C≤12)), substituted alkyl_((C≤12)), or substitutedcycloalkyl_((C≤12)); R₃ is a group of the formula:

wherein: n_(x) is 1-10; X₁, X₂, and X₃ are each independently selectedfrom hydrogen, alkyl_((C≤12)), cycloalkyl_((C≤12)), substitutedalkyl_((C≤12)), or substituted cycloalkyl_((C≤12)); and X₄ and X₅ areeach independently selected from alkyl_((C≤12)), cycloalkyl_((C≤12)),aryl_((C≤12)), heteroaryl_((C≤12)) or a substituted version of any ofthese groups, or X₄ and X₅ are taken together and arealkanediyl_((C≤12)), alkoxydiyl_((C≤12)), alkylaminodiyl_((C≤12)), or asubstituted version of any of these groups; x is an integer from 1 to150; R₄ is a group of the formula:

wherein: n_(y) is 1-10; X₁′, X₂′, and X₃′ are each independentlyselected from hydrogen, alkyl_((C≤12)), cycloalkyl_((C≤12)), substitutedalkyl_((C≤12)), or substituted cycloalkyl_((C≤12)); and X₄′ and X_(S)′are each independently selected from alkyl_((C≤12)),cycloalkyl_((C≤12)), aryl_((C≤12)), heteroaryl_((C≤12)) or a substitutedversion of any of these groups, or X₄′ and X₅′ are taken together andare alkanediyl_((C≤12)), alkoxydiyl_((C≤12)), alkylaminodiyl_((C≤12)),or a substituted version of any of these groups; y is an integer from 1to 150; R₅ is a group of the formula:

wherein: n_(z) is 1-10; Y₁, Y₂, and Y₃ are each independently selectedfrom hydrogen, alkyl_((C≤12)), cycloalkyl_((C≤12)), substitutedalkyl_((C≤12)), or substituted cycloalkyl_((C≤12)); and Y₄ is hydrogen,alkyl_((C≤12)), acyl_((C≤12)), substituted alkyl_((C≤12)), substitutedacyl_((C≤12)), a dye, or a fluorescence quencher; z is an integer from0-6; and R₆ is hydrogen, halo, hydroxy, alkyl_((C≤12)), or substitutedalkyl_((C≤12)), wherein R₃, R₄, and R₅ can occur in any order within thepolymer, provided that R₃ and R₄ are not the same group. In someembodiments, the compound is further defined by the formula wherein: R₁is hydrogen, alkyl_((C≤12)), substituted alkyl_((C≤12)), or

or a metal chelating group; n is an integer from 10 to 500; R₂ and R₂′are each independently selected from hydrogen, alkyl_((C≤12)), orsubstituted alkyl_((C≤12)); R₃ is a group of the formula:

wherein: X₁, X₂, and X₃ are each independently selected from hydrogen,alkyl_((C≤12)), or substituted alkyl_((C≤12)); and X₄ and X₅ are eachindependently selected from alkyl_((C≤12)), aryl_((C≤12)),heteroaryl_((C≤12)) or a substituted version of any of these groups, orX₄ and X₅ are taken together and are alkanediyl_((C≤8)),alkoxydiyl_((C≤8)), alkylaminodiyl_((C≤8)), or a substituted version ofany of these groups; x is an integer from 1 to 100; R₄ is a group of theformula:

wherein: X₁′, X₂′, and X₃′ are each independently selected fromhydrogen, alkyl_((C≤12)), or substituted alkyl_((C≤12)); and X₄′ and X₅′are each independently selected from alkyl_((C≤12)), aryl_((C≤12)),heteroaryl_((C≤12)) or a substituted version of any of these groups, orX₄′ and X₅′ are taken together and are alkanediyl_((C≤8)),alkoxydiyl_((C≤8)), alkylaminodiyl_((C≤8)), or a substituted version ofany of these groups; y is an integer from 1 to 100; R₅ is a group of theformula:

wherein: Y₁, Y₂, and Y₃ are each independently selected from hydrogen,alkyl_((C≤12)), substituted alkyl_((C≤12)); and Y₄ is hydrogen,acyl_((C≤12)), substituted acyl_((C≤12)), a dye, or a fluorescencequencher; z is an integer from 0-6; and R₆ is hydrogen, halo,alkyl_((C≤12)), or substituted alkyl_((C≤12)), wherein R₃, R₄, and R₅can occur in any order within the polymer, provided that R₃ and R₄ arenot the same group. In some embodiments, the compound is further definedby the formula wherein: R₁ is hydrogen, alkyl_((C≤8)), substitutedalkyl_((C≤8)), or

or a metal chelating group; n is an integer from 10 to 200; R₂ and R₂′are each independently selected from hydrogen, alkyl_((C≤8)), orsubstituted alkyl_((C≤8)); R₃ is a group of the formula:

wherein: X₁, X₂, and X₃ are each independently selected from hydrogen,alkyl_((C≤8)), or substituted alkyl_((C≤8)); and X₄ and X₅ are eachindependently selected from alkyl_((C≤12)), aryl_((C≤12)),heteroaryl_((C≤12)) or a substituted version of any of these groups, orX₄ and X₅ are taken together and are alkanediyl_((C≤8)) or substitutedalkanediyl_((C≤8)); x is an integer from 1 to 100; R₄ is a group of theformula:

wherein: X₁′, X₂′, and X₃′ are each independently selected fromhydrogen, alkyl_((C≤8)), or substituted alkyl_((C≤8)); and X₄′ and X₅′are each independently selected from alkyl_((C≤12)), aryl_((C≤12)),heteroaryl_((C≤12)) or a substituted version of any of these groups, orX₄′ and X₅′ are taken together and are alkanediyl_((C≤8)) or substitutedalkanediyl_((C≤8)); y is an integer from 1 to 100; R₅ is a group of theformula:

wherein: Y₁, Y₂, and Y₃ are each independently selected from hydrogen,alkyl_((C≤8)), substituted alkyl_((C≤8)); and Y₄ is hydrogen, a dye, ora fluorescence quencher; z is an integer from 0-6; and R₆ is hydrogen,halo, alkyl_((C≤6)), or substituted alkyl_((C≤6)), wherein R₃, R₄, andR₅ can occur in any order within the polymer, provided that R₃ and R₄are not the same group. In some embodiments, R₁ is hydrogen. In someembodiments, R₁ is alkyl_((C≤6)). In some embodiments, R₁ is methyl. Insome embodiments, R₁ is

In some embodiments, R₁ is a metal chelating group such as a metalchelating group selected from1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, (DOTA),1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid, (TETA),1,8-Diamino-3,6,10,13,16,19-hexaazabicyclo[6,6,6]-eicosane (Diamsar),1,4,7-triazacyclononane-1,4,7-triacetic acid, (NOTA),{4-[2-(bis-carboxymethylamino)-ethyl]-7-carboxymethyl-[1,4,7]triazonan-1-yl}-aceticacid (NETA), N,N′,N″, tris(2-mercaptoethyl)-1,4,7-triazacyclononane(TACN-TM), diethylenetriaminepentaacetic acid (DTPA),1,4,7-triazacyclononane-1,4,7-tris[methyl(2-carboxyethyl)phosphinicacid] (TRAP),1,4,7-triazacyclononane-1,4-bis[methylene(hydroxymethyl)phosphinicacid]-7-[methylene(2-carboxyethyl)phosphinic acid] (NOPO),1,4-bis(carboxymethyl)-6-[bis(carboxymethyl)]amino-6-methylperhydro-1,4-diazepine(AAZTA), 2,2′-(6-((carboxymethyl)amino)-1,4-diazepane-1,4-diyl)diaceticacid (DATA), N,N′-bis(2-hydroxybenzyl)-ethylenediamine-N,N0-diaceticacid, (HBED),N,N′-bis(2-hydroxy-5-sulfobenzyl)-ethylenediamine-N,N′-diacetic acid(SHBED), bis(2-pyridylcarbonyl) amine (BPCA),4-acetylamino-4-[2-[(3-hydroxy-1,6-dimethyl-4-oxo-1,4-dihydro-pyridin-2-ylmethyl)-carbamoyl]-ethyl]-heptanedioicacidbis-[(3-hydroxy-1,6-dimethyl-4-oxo-1,4-dihydro-pyridin-2-ylmethyl)-amide](CP256), desferrioxamine B (DFO),3,6,9,15-tetraazabicyclo[9.3.1]-pentadeca-1(15),11,13-triene-3,6,9,-triacetic acid (PCTA),1,4,7,10,13,16-hexaazacyclohexadecane-N,N′,N″,N′″,N″″,N″″-hexaaceticacid (HEHA),1,4,7,10,13-pentaazacyclopentadecane-N,N′,N″,N′″,N″″-pentaacetic acid(PEPA), or a derivative thereof. In some embodiments, the metalchelating group is a nitrogen containing macrocycle. In someembodiments, the nitrogen containing macrocycle is a compound of theformula:

wherein:

-   -   R₇, R₈, R₉, R₁₀, R₇′, R₈′, and R₉′ are each independently        selected from hydrogen, alkyl_((C≤12)), acyl_((C≤12)),        -alkanediyl_((C≤12))-acyl_((C≤12)), or a substituted version of        any of these groups; or a linker, wherein the linker is an        alkanediyl_((C≤12))-C(O)NH— or a substituted        alkanediyl_((C≤12))-C(O)NH—; or    -   R₇ is taken together with one of R₈, R₉, or R₁₀ and is        alkanediyl_((C≤6)); or    -   R₈ is taken together with one of R₇, R₉, or R₁₀ and is        alkanediyl_((C≤6)); or    -   R₉ is taken together with one of R₇, R₈, or R₁₀ and is        alkanediyl_((C≤6)); or    -   R₁₀ is taken together with one of R₇, R₈, or R₉ and is        alkanediyl_((C≤6)); or    -   R₇′ is taken together with one of R₈′ or R₉′ and is        alkanediyl_((C≤6)); or    -   R₈′ is taken together with one of R₇′ or R₉′ and is        alkanediyl_((C≤6)); or    -   R₉′ is taken together with one of R₇′ or R₈′ and is        alkanediyl_((C≤6)); and    -   a, b, c, d, a′, b′, and c′ are each independently selected from        1, 2, 3, or 4.

In some embodiments, a, b, c, d, a′, b′, and c′ are each independentlyselected from 2 or 3. In some embodiments, the metal chelating group is:

In some embodiments, the metal chelating complex is bound to a metalion. In some embodiments, the metal ion is a radionuclide or radiometal.In some embodiments, the metal ion is suitable for PET or SPECT imaging.In some embodiments, the metal chelating complex is bound to atransition metal ion. In some embodiments, the metal ion is a copperion, a gallium ion, a scandium ion, an indium ion, a lutetium ion, aytterbium ion, a zirconium ion, a bismuth ion, a lead ion, a actiniumion, or a technetium ion. In some embodiments, the metal ion is anisotope selected from ^(99m)Tc, ⁶⁰Cu, ⁶¹Cu, ⁶²Cu, ⁶⁴Cu, ⁸⁶Y, ⁹⁰y, ⁸⁹Zr,⁴Sc, ⁴⁷Sc, ⁶⁶Ga, ⁶⁷Ga, ⁶⁸Ga, ¹¹¹In, ¹⁷⁷Lu, ²²⁵Ac, ²¹²Pb, ²¹²Bi, ²¹³Bi,¹¹¹In, ^(114m)In, ¹¹⁴In, ¹⁸⁶Re, or ¹⁸⁸Re. In some embodiments, thetransition metal is a copper(II) ion. In some embodiments, thecopper(II) ion is a ⁶⁴Cu²⁺ ion. In some embodiments, the metal chelatingcomplex is:

In some embodiments, R₂ is alkyl_((C≤6)). In some embodiments, R₂ ismethyl. In some embodiments, R₂′ is alkyl_((C≤6)). In some embodiments,R₂′ is methyl. In some embodiments, R₃ is further defined by theformula:

wherein: X₁ is selected from hydrogen, alkyl_((C≤8)), or substitutedalkyl_((C≤8)); and X₄ and X₅ are each independently selected fromalkyl_((C≤12)), aryl_((C≤12)), heteroaryl_((C≤12)) or a substitutedversion of any of these groups, or X₄ and X₅ are taken together and arealkanediyl_((C≤8)) or substituted alkanediyl_((C≤8)); In someembodiments, X₁ is alkyl_((C≤6)). In some embodiments, X₁ is methyl. Insome embodiments, X₄ is alkyl_((C≤8)). In some embodiments, X₄ ismethyl, ethyl, propyl, butyl, or pentyl. In some embodiments, X₅ isalkyl_((C≤8)). In some embodiments, X₅ is methyl, ethyl, propyl, butyl,or pentyl.

In some embodiments, R₄ is further defined by the formula:

wherein: X₁′ is selected from hydrogen, alkyl_((C≤8)), or substitutedalkyl_((C≤8)); and X₄′ and X₅′ are each independently selected fromalkyl_((C≤12)), aryl_((C≤12)), heteroaryl_((C≤12)) or a substitutedversion of any of these groups, or X₄′ and X₅′ are taken together andare alkanediyl_((C≤8)) or substituted alkanediyl_((C≤8)). In someembodiments, X₁′ is alkyl_((C≤6)). In some embodiments, X₁ is methyl. Insome embodiments, X₄′ is alkyl_((C≤8)). In some embodiments, X₄′ ismethyl, ethyl, propyl, butyl, or pentyl. In some embodiments, X₅′ isalkyl_((C≤8)). In some embodiments, X₅′ is methyl, ethyl, propyl, butyl,or pentyl. In some embodiments, R₅ is further defined by the formula:

wherein: Y₁ is selected from hydrogen, alkyl_((C≤8)), substitutedalkyl_((C≤8)); and Y₄ is hydrogen, a dye, or a fluorescence quencher. Insome embodiments, Y₁ is alkyl_((C≤6)). In some embodiments, Y₁ ismethyl. In some embodiments, Y₄ is hydrogen. In some embodiments, Y₄ isa dye. In some embodiments, Y₄ is fluorescent dye. In some embodiments,the fluorescent dye is a coumarin, fluorescein, rhodamine, xanthene,BODIPY® (boron-dipyrromethene), Alexa Fluor® (sulfonated derivative ofcoumarin, rhodamine, xanthene or cyanine dye), or cyanine dye. In someembodiments, the fluorescent dye is indocyanine green, AMCA-x, MarinaBlue, PyMPO, Rhodamine Green™ (rhodamine), Tetramethylrhodamine,5-carboxy-X-rhodamine, Bodipy493, Bodipy TMR-x, Bodipy630, Cyanine5,Cyanine5.5, and Cyanine7.5. In some embodiments, the fluorescent dye isindocyanine green. In some embodiments, Y₄ is a fluorescence quencher.In some embodiments, the fluorescence quencher is QSY7, QSY21, QSY35,BHQ1, BHQ2, BHQ3, TQ1, TQ2, TQ3, TQ4, TQ5, TQ6, and TQ7. In someembodiments, n is 75-150. In some embodiments, n is 100-125. In someembodiments, x is 1-99. In some embodiments, x is from 1-5, 5-10, 10-15,15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65,65-70, 70-75, 75-80, 80-85, 85-90, 90-95, 95-99 or any range derivabletherein. In some embodiments, y is 1-99. In some embodiments, y is from1-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50,50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, 95-99 orany range derivable therein. In some embodiments, z is 0-6. In someembodiments, z is 1-6. In some embodiments, z is from 0-2, 2-4, 4-6, orany range derivable therein. In some embodiments, R₃, R₄, and R₅ canoccur in any order within the polymer. In some embodiments, R₃, R₄, andR₅ occur in the order described in formula I. In some embodiments, thepolymer further comprises a targeting moiety. In some embodiments, thetargeting moiety is a small molecule, an antibody, an antibody fragment,or a signaling peptide. In some embodiments, R₃ and R₄ are selectedfrom:

In some embodiments, the polymer is PEO₁₁₄-P(DEA₂₀-D5A₆₀),PEO₁₁₄-P(DEA₄₀-D5A₄₀), PEO₁₁₄-P(DEA₆₀-D5A₂₀), PEO₁₁₄-P(DPA₆₀-DBA₂₀),PEO₁₁₄-P(DPA₄₀-DBA₄₀), PEO₁₁₄-P(DPA₂₀-DBA₆₀), PEO₁₁₄-P(DEA₇₆-DPA₂₄),PEO₁₁₄-P(DEA₅₈-DPA₄₂), PEO₁₁₄-P(DEA₃₉-DPA₆₁), PEO₁₁₄-P(DEA₂₁-DPA₇₉),PEO₁₁₄-P(DPA₃₀-DBA₅₀), PEO₁₁₄-P(DBA₂₈-D5A₅₂), PEO₁₁₄-P(DBA₅₆-D5A₂₄),PEO₁₁₄-P(DEA₂₀-D5A₆₀-AMA₃), PEO₁₁₄-P(DEA₄₀-D5A₄₀-AMA₃),PEO₁₁₄-P(DEA₆₀-D5A₂₀-AMA₃), PEO₁₁₄-P(DPA₆₀-DBA₂₀-AMA₃),PEO₁₁₄-P(DPA₄₀-DBA₄₀-AMA₃), PEO₁₁₄-P(DPA₂₀-DBA₆₀-AMA₃),PEO₁₁₄-P(DEA₇₆-DPA₂₄-AMA₃), PEO₁₁₄-P(DEA₅₈-DPA₄₂-AMA₃),PEO₁₁₄-P(DEA₃₉-DPA₆₁-AMA₃), PEO₁₁₄-P(DEA₂₁-DPA₇₉-AMA₃),PEO₁₁₄-P(DPA₃₀-DBA₅₀-AMA₃), PEO₁₁₄-P(DBA₂s-D5A₅₂-AMA₃), orPEO₁₁₄-P(DBA₅₆-D5A₂₄-AMA₃), PEO₁₁₄-P(DEA₁₁-EPA₈₉),PEO₁₁₄-P(DEA₂₂-EPA₇₈), PEO₁₁₄-P(EPA₉₀-DPA₁₀), PEO₁₁₄-P(EPA₇₉-DPA₂₁);wherein PEO is polyethylene glycol; P is poly; DBA is2-(dibutylamino)ethyl methacrylate; D5A is 2-(dipentylamino)ethylmethacrylate; AMA is 2-aminoethyl methacrylate; DEA is2-(diethylamino)ethyl methacrylate; DPA is 2-(dipropylamino)ethylmethacrylate; and EPA is 2-(ethylpropylamino)ethyl methacrylate.

In another aspect, the present disclosure provides a polymer of theformula:

wherein: R₁ is hydrogen, alkyl_((C≤12)), cycloalkyl_((C≤12)),substituted alkyl_((C≤12)), substituted cycloalkyl_((C≤12)), or

or a metal chelating group; n is an integer from 1 to 500; R₂ and R₂′are each independently selected from hydrogen, alkyl_((C≤12)),cycloalkyl_((C≤12)), substituted alkyl_((C≤12)), or substitutedcycloalkyl_((C≤12)); R₃ is a group of the formula:

wherein: n_(x) is 1-10; X₁, X₂, and X₃ are each independently selectedfrom hydrogen, alkyl_((C≤12)), cycloalkyl_((C≤12)), substitutedalkyl_((C≤12)), or substituted cycloalkyl_((C≤12)); X₄ is pentyl,n-propyl, or ethyl; and X₅ is pentyl or n-propyl; x is an integer from 1to 100; R₄ is a group of the formula:

wherein: Y₁, Y₂, and Y₃ are each independently selected from hydrogen,alkyl_((C≤12)), cycloalkyl_((C≤12)), substituted alkyl_((C≤12)), orsubstituted cycloalkyl_((C≤12)); and Y₄ is hydrogen, alkyl_((C≤12)),acyl_((C≤12)), substituted alkyl_((C≤12)), substituted acyl_((C≤12)), adye, or a fluorescence quencher; y is an integer from 1 to 6; and R₅ ishydrogen, halo, hydroxy, alkyl_((C≤12)), or substituted alkyl_((C≤12)).In some embodiments, Y₄ is a fluorescent dye. In some embodiments, thefluorescent dye is indocyanine green. In some embodiments, the polymeris PEO₁₁₄-P(D5A₈₀), PEO₁₁₄-P(D5A₁₀₀), PEO₁₁₄-P(DPA₈₀), PEO₁₁₄-P(DPA₁₀₀),PEO₁₁₄-P(EPA₈₀), and PEO₁₁₄-P(EPA₁₀₀); wherein PEO is polyethyleneglycol; P is poly; D5A is 2-(dipentylamino)ethyl methacrylate; DPA is2-(dipropylamino)ethyl methacrylate; and EPA is2-(ethylpropylamino)ethyl methacrylate.

In yet another aspect, the present disclosure provides a compound of theformula:

In another aspect, the present disclosure provides a micelle of apolymer of the present disclosure.

In yet another aspect, the present disclosure provides a pH responsivesystem comprising a micelle of a first polymer of the presentdisclosure, wherein z is not 0 and Y₄ is a dye, and wherein the micellehas a pH transition point and an emission spectra. In some embodiments,the micelle further comprises a second polymer of the presentdisclosure, wherein z is not 0 and Y₄ is a fluorescence quencher. Insome embodiments, the second polymer has the same formula as the firstpolymer except that Y₄ is a fluorescence quencher. In some embodiments,the pH transition point is between 3-9. In some embodiments, the pHtransition point is between 4-8. In some embodiments, the pH transitionpoint is between 4-6. In some embodiments, the pH transition point isbetween 6-7.5. In some embodiments, the pH transition point is 4.38,4.67, 4.96, 5.27, 5.63, 5.91, 6.21, 6.45, 6.76, 7.08, or 7.44. In someembodiments, the emission spectra is between 400-850 nm. In someembodiments, the system has a pH response (ΔpH₁₀₋₉₀%) of less than 1 pHunit. In some embodiments, the pH response is less than 0.25 pH units.In some embodiments, the pH response is less than 0.15 pH units. In someembodiments, the fluorescence signal has a fluorescence activation ratioof greater than 25. In some embodiments, the fluorescence activationratio is greater than 50.

In yet another aspect, the present disclosure provides a method ofimaging the pH of a intracellular or extracellular environmentcomprising:

-   -   (a) contacting a micelle of the present disclosure with the        environment; and    -   (b) detecting one or more optical signals from the environment,        wherein the detection of the optical signal indicates that the        micelle has reached its pH transition point and disassociated.        In some embodiments, the optical signal is a fluorescent signal.        In some embodiments, when the intracellular environment is        imaged, the cell is contacted with the micelle under conditions        suitable to cause uptake of the micelle. In some embodiments,        the intracellular environment is part of a cell. In some        embodiments, the part of the cell is lysosome or an endosome. In        some embodiments, the extracellular environment is of a tumor or        vascular cell. In some embodiments, the extracellular        environment is intravascular or extravascular. In some        embodiments, the imaging the pH of the tumor environment        comprises imaging the sentinel lymph node or nodes. In some        embodiments, imaging sentinel lymph node or nodes allows for the        surgical resection of the tumor and staging of the tumor        metastasis. In some embodiments, imaging the pH of the tumor        environment allows determination of the tumor size and margins.        In some embodiments, imaging the pH of the tumor environment        allows for more precise removal of the tumor during surgery. In        some embodiments, imaging the pH of the sentential lymph node or        nodes allows for more precise removal of the sentential lymph        node or nodes during surgery. In some embodiments, the method        further comprises:    -   (a) contacting the cell with a compound of interest;    -   (b) detecting one or more optical signals in the environment;        and    -   (c) determining whether a change in the optical signal occurred        following contacting the cell with the compound of interest.        In some embodiments, the compound of interest is a drug,        antibody, peptide, protein, nucleic acid, or small molecule.

In yet another aspect, the present disclosure provides a method ofdelivering a compound of interest to a target cell comprising:

-   -   (a) encapsulating the compound of interest with a micelle of a        polymer of claims 1-55; and    -   (b) contacting the target cell with the micelle under such        conditions that the pH of the target cell triggers the        disassociation of the micelle and release of the compound,        thereby delivering the compound of interest.        In some embodiments, the compound of interest is delivered into        the cell. In some embodiments, the compound of interest is        delivered to the cell. In some embodiments, the compound of        interest is a drug, antibody, peptide, protein, nucleic acid, or        small molecule. In some embodiments, the method comprises        administering the micelle to a patient.

In still yet another aspect, the present disclosure provides method ofresecting a tumor in a patient comprising:

-   -   (a) administering to the patient an effective dose of a pH        responsive system of the present disclosure;    -   (b) detecting one or more optical signals for the patient;        wherein the optical signals indicate the presence of a tumor;        and    -   (c) resecting the tumor via surgery.

In some embodiments, the optical signals indicate the margins of thetumor. In some embodiments, the tumor is 90% resected, or the tumor is95% resected., or the tumor is 99% resected. In some embodiments, thetumor is a solid tumor such as a solid tumor is from a cancer. In someembodiments, the cancer is a breast cancer or a head and neck cancersuch as a head and neck squamous cell carcinoma. In some embodiments,the pH responsive system is comprised of a polymer of the formula:

wherein: x is an integer from 30 to 150, y is an integer from 1 or 2; xand y are randomly distributed throughout the polymer; and ICG is thefluorescent dye indocyanine green.

In yet another aspect, the present disclosure provides methods oftreating a cancer susceptible to endosomal/lysosomal pH arrest in apatient comprising administering to the patient in need thereof a pHresponsive system of the present disclosure. In some embodiments, thecancer is a lung cancer such as a non-small cell lung cancer. In someembodiments, the cancer comprises a mutation in the KRAS gene or amutation in the LKB1 gene. In other embodiments, the cancer comprises amutation in both the KRAS and LKB1 gene. In some embodiments, methodsare sufficient to induce apoptosis.

In still yet another aspect, the present disclosure provides methods ofidentifying the presence of a genetic mutation in a cell:

-   -   (a) contacting a pH responsive systems comprising two or more        micelles with the cell or cellular environment; and    -   (b) detecting two or more optical signals from the environment,        wherein the detection of the optical signal indicates that one        of the micelles has reached its pH transition point and        disassociated; and    -   (c) correlate the two or more optical signals to determine the        presence of the genetic mutation in the cell.        In some embodiments, the genetic mutation is a mutation in the        KRAS gene. In some embodiments, the two or more micelles        comprises three micelles with a pH transition point at 6.9, 6.2,        and 5.3. In some embodiments, each of the three micelles is        prepared from a polymer selected from poly(2-dipropylaminoethyl        methacrylate)-tetramethyl rhodamine (PDPA-TMR),        poly((2-ethylpropylamino)ethyl methacrylate)-BODIPY 493        (PEPA-BDY493; wherein BDY is BODIPY), and        poly((2-dibutylamino)ethyl methacrylate)-Cyanine 5(PDBA-Cy5). In        some embodiments, the method is performed in vivo and contact a        cell comprising administering the one or more micelles to a        patient.

In still yet another aspect, the present disclosure provides methods ofidentifying the tumor acidosis pathway comprising:

-   -   (a) contacting a pH responsive system of the present disclosure        comprising one or more micelles with a cell or a cellular        environment;    -   (b) contacting the cell with an inhibitor of the pH regulatory        pathway;    -   (c) detecting two or more optical signals from the cell or        cellular environment, wherein the detection of the optical        signal indicates that one of the micelles has reached its pH        transition point and disassociated; and    -   (d) correlating the two or more optical signals with a        modification in the tumor acidosis pathway.

In some embodiments, the inhibitor of the pH regulatory pathway is aninhibitor of a monocarboxylate transporter, a carbonic anhydrase, ananion exchanger, a Na⁺-bicarbonate exchanger, a Na⁺/H⁺ exchanger, or aV-ATPase. In some embodiments, the one or more micelles comprise apolymer with two or more fluorophores attached to the polymer backbone.In some embodiments, the method comprises one micelle and the micellecomprises two or more polymers with different fluorophores or differentR₃ groups. In some embodiments, the micelle comprises two or morepolymers with different fluorophores and different R₃ groups.

140. A method of imaging a patient to determine the presence of a tumorcomprising:

-   -   (a) contacting a pH responsive system comprising one or more        micelles of the present disclosure with the tumor, wherein the        micelle further comprises a metal chelating group at R₁;    -   (b) collecting one or more PET or SPECT imaging scans; and    -   (c) collecting one or more optical imaging scans, wherein the        detection of the optical signal indicates that one of the        micelles has reached its pH transition point and disassociated;    -   wherein the one or more PET or SPECT imaging scans and the one        or more optical imaging scans result in the identification of a        tumor.

In some embodiments, the optical imaging scans are collected before thePET or SPECT imaging scans. In other embodiments, the optical imagingscans are collected after the PET or SPECT imaging scans. In otherembodiments, the optical imaging scans are collected simultaneously withthe PET or SPECT imaging scans. In some embodiments, the imaging scansare PET imaging scans. In other embodiments, the imaging scans are SPECTimaging scans. In some embodiments, the metal chelating group is boundto a ⁶⁴Cu ion. In some embodiments, the metal chelating group is anitrogen containing macrocycle. In some embodiments, the nitrogencontaining macrocycle is:

wherein: R₇, R₈, R₉, R₁₀, R₇′, R₈′, R₉′ a, b, c, d, a′, b′, and c′ areas defined above. In some embodiments, the nitrogen containingmacrocycle is:

In still yet another aspect, the present disclosure provides polymers ofthe formula:

wherein:

-   -   R₁ is a metal chelating group;    -   n is an integer from 1 to 500;    -   R₂ and R₂′ are each independently selected from hydrogen,        alkyl_((C≤12)), cycloalkyl_((C≤12)), substituted alkyl_((C≤12)),        or substituted cycloalkyl_((C≤12));    -   R₃ is a group of the formula:

-   -   wherein:        -   n_(x) is 1-10;        -   X₁, X₂, and X₃ are each independently selected from            hydrogen, alkyl_((C≤12)), cycloalkyl_((C≤12)), substituted            alkyl_((C≤12)), or substituted cycloalkyl_((C≤12)); and        -   X₄ and X₅ are each independently selected from            alkyl_((C≤12)), cycloalkyl_((C≤12)), aryl_((C≤12)),            heteroaryl_((C≤12)) or a substituted version of any of these            groups, or X₄ and X₅ are taken together and are            alkanediyl_((C≤12)), alkoxydiyl_((C≤12)),            alkylaminodiyl_((C≤12)), or a substituted version of any of            these groups;    -   x is an integer from 1 to 150;    -   R₄ is a group of the formula:

-   -   wherein:        -   n_(z) is 1-10;        -   Y₁, Y₂, and Y₃ are each independently selected from            hydrogen, alkyl_((C≤12)), cycloalkyl_((C≤12)), substituted            alkyl_((C≤12)), or substituted cycloalkyl_((C≤12)); and        -   Y₄ is hydrogen, alkyl_((C≤12)), acyl_((C≤12)), substituted            alkyl_((C≤12)), substituted acyl_((C≤12)), a dye, or a            fluorescence quencher;    -   y is an integer from 0-6; and    -   R₄ is hydrogen, halo, hydroxy, alkyl_((C≤12)), or substituted        alkyl_((C≤12)),    -   wherein R₃ and R₄ can occur in any order within the polymer,        provided that R₃ and R₄ are not the same group.

In some embodiments, R₃ is:

wherein: X₁, X₄, and X₅ are as defined above.

In some embodiments, X₁ is alkyl_((C≤12)) such as X₁ is methyl. In someembodiments, X₄ and X₅ are taken together and are alkanediyl_((C≤12)) orsubstituted alkanediyl_((C≤12)). In some embodiments, wherein X₄ and X₅are taken together and are —CH₂CH₂CH₂CH₂CH₂CH₂—. In some embodiments, R₄is:

wherein: Y₁ and Y₄ are as defined above. In some embodiments, Y₄ is adye. In some embodiments, Y₄ is a fluorescent dye. In some embodiments,Y₁ is alkyl_((C≤12)) such as Y₁ is methyl. In some embodiments, x is 40,60, 80, 100, or 120. In some embodiments, y is 1, 2, or 3, such as wheny is 3. In some embodiments, the polymer is PEO₁₁₄-P(C7A₄₀-r-ICG₃),PEO₁₁₄-P(C7A₆₀-r-ICG₃), PEO₁₁₄-P(C7A₈₀-r-ICG₃), PEO₁₁₄-P(C7A₁₀₀-r-ICG₃),or PEO₁₁₄-P(C7A₁₂₀-r-ICG₃), wherein the PEO group is capped with a metalchelating group; PEO is polyethylene glycol; P is poly; C7A is2-(hexamethyleneimino)ethyl methacrylate; ICG is indocyanine green; andr is for describing that the arrangement of the two monomeric units thatare connected is random.

As used herein, “pH responsive system,” “micelle,” “pH-responsivemicelle,” “pH-sensitive micelle,” “pH-activatable micelle” and“pH-activatable micellar (pHAM) nanoparticle” are used interchangeablyherein to indicate a micelle comprising one or more block copolymers,which disassociates depending on the pH (e.g., above or below a certainpH). As a non-limiting example, at a certain pH, the block copolymer issubstantially in micellar form. As the pH changes (e.g., decreases), themicelles begin to disassociate, and as the pH further changes (e.g.,further decreases), the block copolymer is present substantially indisassociated (non-micellar) form.

As used herein, “pH transition range” indicates the pH range over whichthe micelles disassociate.

As used herein, “pH transition value” (pH_(t)) indicates the pH at whichhalf of the micelles are disassociated.

It is contemplated that any method or composition described herein canbe implemented with respect to any other method or composition describedherein.

The terms “comprise” (and any form of comprise, such as “comprises” and“comprising”), “have” (and any form of have, such as “has” and“having”), “contain” (and any form of contain, such as “contains” and“containing”), and “include” (and any form of include, such as“includes” and “including”) are open-ended linking verbs. As a result, amethod, composition, kit, or system that “comprises,” “has,” “contains,”or “includes” one or more recited steps or elements possesses thoserecited steps or elements, but is not limited to possessing only thosesteps or elements; it may possess (i.e., cover) elements or steps thatare not recited. Likewise, an element of a method, composition, kit, orsystem that “comprises,” “has,” “contains,” or “includes” one or morerecited features possesses those features, but is not limited topossessing only those features; it may possess features that are notrecited.

Any embodiment of any of the present methods, composition, kit, andsystems may consist of or consist essentially of—rather thancomprise/include/contain/have—the described steps and/or features. Thus,in any of the claims, the term “consisting of” or “consistingessentially of” may be substituted for any of the open-ended linkingverbs recited above, in order to change the scope of a given claim fromwhat it would otherwise be using the open-ended linking verb.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.” Throughout thisapplication, the term “about” is used to indicate that a value includesthe standard deviation of error for the device or method being employedto determine the value. Following long-standing patent law, the words“a” and “an,” when used in conjunction with the word “comprising” in theclaims or specification, denotes one or more, unless specifically noted.

Other objects, features and advantages of the present disclosure willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the disclosure, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the disclosure will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentdisclosure. The disclosure may be better understood by reference to oneor more of these drawings in combination with the detailed descriptionof specific embodiments presented herein.

FIG. 1 shows the syntheses of dye- or fluorescence quencher(FQ)-conjugated PEO-b-P(R₁-r-R₂) copolymers. The hydrophobicity of thePR segment can be continuously controlled by varying the molar fractionsof the two monomers (R₁ or R₂: Et, ethyl; Pr, propyl; Bu, butyl; Pe,pentyl).

FIG. 2 shows the ¹H NMR spectra of PEO-P(DEA_(x)-D5A_(y)) (x+y=80)copolymers at different monomer (DEA and D5A) ratios in the randomcopolymers. The peaks at 0.9 ppm and 1.1 ppm were used to estimate themonomer composition in the hydrophobic PR block.

FIG. 3 shows the ¹H NMR spectra of PEO-P(DPA_(x)-DBA_(y)) (x+y=80)copolymers at different monomer (DPA and DBA) ratios in the randomcopolymers. The peaks at 1.3 ppm and 1.4 ppm were used to estimate themonomer composition in the hydrophobic PR block.

FIG. 4 shows the ¹H NMR spectra of nanoprobe compositions with pH_(t)values at 7.8, 7.4, 7.1, 6.8, 6.5 and 6.2 by adjusting the monomer (DEAand DPA) ratios in the hydrophobic PR block. The peaks at 0.9 ppm and1.0-1.1 ppm were used to estimate the monomer composition in thehydrophobic PR block.

FIG. 5 shows the ¹H NMR spectra of nanoprobe compositions with pH_(t)values at 6.2, 5.9, 5.6 and 5.3 by adjusting the monomer (DPA and DBA)ratios in the hydrophobic PR block. The peaks at 1.3 ppm and 1.4 ppmwere used to estimate the monomer composition in the hydrophobic PRblock.

FIG. 6 shows the ¹H NMR spectra of nanoprobe compositions with pH_(t)values at 5.3, 5.0, 4.7 and 4.4 by adjusting the monomer (DBA and D5A)ratios in the hydrophobic PR block. The peaks at 1.3 ppm and 1.4 ppmwere used to estimate the monomer composition in the hydrophobic PRblock.

FIGS. 7A-C: (FIG. 7A) Comparison of PDEA/PD5A molecular mixture vs.P(DEA₄₀-D5A₄₀) copolymer strategies for the control of pH_(t). (FIG. 7B)Normalized fluorescence intensity of P(DEA_(x)-D5A_(y)) nanoprobes withdifferent ratios of the two monomers as a function of pH. (FIG. 7C)Nanoprobe pH_(t) is linearly correlated with the molar fraction of theDEA-MA monomer in the PR segment. Polymer concentrations were 0.1 mg/mLin these studies.

FIG. 8 shows the pH-dependent fluorescence spectra of PDEA/PD5A micelleblend vs. P(DEA₄₀-D5A₄₀) copolymer nanoprobes. Cy5 dye(λ_(ex)/λ_(em)=646/662 nm) was conjugated to the PR blocks of thecorresponding copolymers. The normalized fluorescence intensity vs. pHrelationships were shown in FIG. 7A.

FIG. 9 shows pH-dependent fluorescence spectra of coarse-tunedP(DEA_(x)-D5A_(y)) nanoprobes. Cy5 dye (λ_(ex)/λ_(em)=646/662 nm) wasconjugated to the PR blocks of the copolymers. The normalizedfluorescence intensity vs. pH relationships were shown in FIG. 7B.

FIGS. 10A-D: (FIG. 10A) Normalized fluorescence intensity as a functionof pH for Cy5-conjugated P(DPA_(x)-DBA_(y)) nanoprobes. (FIG. 10B) Aderivatized fluorescence plot (d_(F)/d_(pH), data from 4a) as a functionof pH for P(DPA_(x)-DBA_(y)) vs. P(DEA₄₀-D5A₄₀) nanoprobes. Use ofmethacrylate monomers with close hydrophobicity (i.e., DPA/DBA vs.DEA/D5A) resulted in much sharper pH transitions. (FIG. 10C) Linearrelationships of the nanoprobe pH_(t) as a function of molar fractionsof the less hydrophobic monomer for different copolymer compositions.These correlations serve as the standard curves for selecting theoptimal copolymer composition to achieve an operator-predeterminedpH_(t). (FIG. 10D) A representative library of UPS nanoprobes with 0.3pH increment covering the entire physiologic range of pH (4-7.4). Allthe nanoprobes were conjugated with the Cy5 dye. Polymer concentrationswere at 0.1 mg/mL.

FIG. 11 shows pH-dependent fluorescence spectra of fine-tunedP(DPA_(x)-DBA_(y)) nanoprobes. Cy5 dye (λ_(ex)/λ_(em)=646/662 nm) wasconjugated to the PR blocks of the copolymers. The normalizedfluorescence intensity vs. pH relationships were shown in FIG. 10A.

FIG. 12 shows pH-dependent fluorescence spectra of the UPS librarynanoprobes. The composition for each UPS nanoprobe is shown in Table 3.Cy5 dye (λ_(ex)/λ_(em)=646/662 nm) was conjugated to the PR blocks ofthe copolymers. The normalized fluorescence intensity vs. pHrelationships were shown in FIG. 10D.

FIG. 13 shows the fluorescence imaging of the UPS library consisting of10 nanoprobes with 0.3 pH increment. The composition for each UPSnanoprobe is shown in Table 3. Cy5 dye (λ_(ex)/λ_(em)=646/662 nm) wasconjugated to the PR blocks of the copolymers. Images of the nanoprobeswere taken on a Maestro Imaging system.

FIGS. 14A-F show (FIG. 14A) Structures of the PEO-PDPA-Dye/FQcopolymers. (FIG. 14B) Structures of selected fluorophores with largeStokes shift. (FIG. 14C) Structures of selected Rhodamine dyes. (FIG.14D) Structures of selected Bodipy dyes. (FIG. 14E) Structures ofselected cyanine dyes. The excitation/emission wavelengths for all thefluorophores were shown in FIGS. 14B-E, respectively. (FIG. 14F)Structures of the selected fluorescence quenchers. The active quenchingrange of each quencher was shown in parenthesis.

FIGS. 15A & B show a schematic design of ultra-pH sensitive (UPS)micellar nanoprobes. (FIG. 15A) In the unimer state (pH<pH_(t)), polymerdissociation resulted in fluorophore-quencher separation and strongfluorescence emission. In the micelle state (pH>pH_(t)), fluorescencequenching dramatically suppress the emission intensity of fluorophores.(FIG. 15B) A copolymer strategy was used to achieve anoperator-predetermined control of nanoprobe pH_(t) by the ability tocontinuously fine tuning the hydrophobicity of the PR segment.

FIGS. 16A & B show the fluorescence intensity ratios of mixednanoparticles at pH 5.0 (ON) and pH 7.4 (OFF) at different ratios ofPDPA-Dye/PDPA-FQ. (FIG. 16A) Results for fluorophores with large Stokesshift (AMCA, MB and PPO). (FIG. 16B) Results for BODIPY® families offluorophores. The structures of the fluorophores and FQs were shown inFIGS. 14A-F.

FIGS. 17A-F show the pH-dependent fluorescence spectra of nanoprobeswithout (left column) or with (right column) fluorescence quenchers.Fluorophores with large Stokes shift were presented in this study. Thestructures of the fluorophores and FQs were shown in FIGS. 14A-F.

FIGS. 18A-F show the introduction of FQ-conjugated PDPA copolymersignificantly increased the fluorescence activation ratio of differentPDPA-dye nanoprobes. Fluorescence intensity ratio at different pH to pH7.4 (F_(pH)/F_(7.4)) was plotted for copolymer alone (FIGS. 18A, 18C,and 18E) and with the addition of FQ-conjugated copolymers (FIGS. 18B,18D, and 18F). The structures of the fluorophores and FQs were shown inFIGS. 14A-F.

FIGS. 19A-F show the pH-dependent fluorescence spectra of nanoprobeswithout (left column) or with (right column) fluorescence quenchers.BODIPY® family of fluorophores were presented in this study. Thestructures of the fluorophores and FQs were shown in FIGS. 14A-F.

FIGS. 20A-F show the pH-dependent fluorescence spectra of nanoprobeswithout (left column) or with (right column) fluorescence quenchers.Rhodamine family of fluorophores were presented in this study. Thestructures of the fluorophores and FQs were shown in FIGS. 14A-F.

FIGS. 21A-E show the pH-dependent fluorescence spectra of nanoprobeswithout (left column) or with (right column) fluorescence quenchers.Cyanine family of fluorophores were presented in this study. Thestructures of the fluorophores and FQs were shown in FIGS. 14A-F.

FIGS. 22A-D show the fluorescence intensity ratio at different pH to pH7.4 (F_(pH)/F_(7.4)) was plotted for copolymer alone (FIG. 22A, 22C) andwith the addition of FQ-conjugated copolymers (FIG. 22B, 22D) forrhodamine and cyanine families of dyes. The structures of thefluorophores and FQs were shown in FIGS. 14A-F.

FIGS. 23A & B show Aerobic glycolysis and acidic extracellular pH(pH_(e)) in the tumor. (FIG. 23A) Cancer cells convert glucose mostly tolactate regardless of whether oxygen is present (Warburg effect). Thefigure is adapted from Heiden, et al., 2009. (FIG. 23B) Acidic pH_(e)measured in 269 tumors from 30 different human cancer cell lines. Theaverage pH_(e)=6.84 with variation range from 6.71-7.01, which is belowthe blood pH (7.4). The figure is adapted from Volk, et al., 1993.

FIG. 24 shows an exemplary UPS library consisting of 10 nanoprobesspanning a wide pH range (4-7.4) and large fluorescent emissions(400-820 nm). Each nanoprobe is encoded by its transition pH andfluorophore. Images of 4.4-AMCA and 4.7-MB were taken by a camera at theexcitation light of 365 nm. Images of the rest of the nanoprobesolutions were taken on a Maestro Imaging system.

FIG. 25 shows the self-assembly of ionizable polymeric micelles by twoindependent mechanisms. The left panel shows the induction ofmicellization by pH increase, where the PR segments become neutralizedand hydrophobic to drive micelle formation. Surprisingly, addition ofchaotropic ions (CA, such as ClO₄ ⁻) at low pH also leads tomicellization with ammonium PR segments (right panel). Structures of aseries of PEO-b-PR copolymers (1-5) with different hydrophobic sidechains are shown in the inset.

FIGS. 26A-C show (FIG. 26A) Chaotropic anions induce micelleself-assembly from PEO-b-PR copolymers with protonated PR segment, areversed “salt-out” effect from their abilities to solubilize proteins(salt-in). (FIG. 26B) Illustration of FRET design to investigateCA-induced micelle self-assembly. Addition of CA results in micelleformation and efficient energy transfer from donor (TMR) to acceptor(Cy5) dyes. (FIG. 26C) Chaotropic anion-induced micelle self-assemblyshowing the anti-Hofmeister trend.

FIG. 27 shows the fluorescence spectra of FRET polymer pairs ofPEO-b-P(DPA-r-TMR)/PEO-b-P(DPA-r-Cy5) at different concentrations ofchaotropic anions. The samples were excited at λ_(ex)=545 nm andemission spectra were collected from 560-750 nm. All the experimentswere conducted at pH=4, below the transition pH of PEO-b-PDPA(pH_(t)=6.1).

FIG. 28 shows the fluorescence spectra of FRET polymer pairs ofPEO-b-P(DPA-r-TMR)/PEO-b-P(DPA-r-Cy5) at different concentrations ofkosmotropic and borderline anions. The samples were excited atλ_(ex)=545 nm and emission spectra were collected from 560-750 nm. Allthe experiments were conducted at pH=4, below the transition pH ofPEO-b-PDPA (pH_(t)=6.1).

FIGS. 29A & B show TEM and DLS analyses of micelle transition ofcopolymer 3 in the presence of Cl⁻ (FIG. 29A) and ClO₄ ⁻ anions (FIG.29B). Concentrations of both anions were controlled at 50 mM (pH=5.0).The scale bars are 100 nm in the TEM images.

FIGS. 30A & B show (FIG. 30A) TEM and (FIG. 30B) DLS analyses of micellemorphology and hydrodynamic diameter of copolymer 3 micelles in thepresence of Cl⁻ (50 mM) at pH 7.4. The scale bar is 100 nm in the TEMimage.

FIGS. 31A & B show (FIG. 31A) TEM and (FIG. 31B) DLS analyses of micellemorphology and hydrodynamic diameter of copolymer 3 micelles in thepresence of ClO₄ ⁻ (50 mM) at pH 7.4. The scale bar is 100 nm in the TEMimage.

FIGS. 32A & B show (FIG. 32A) TEM and (FIG. 32B) DLS analyses of micellemorphology and hydrodynamic diameters of PEO-b-PLA copolymer in thepresence of ClO₄ ⁻ (50 mM) at pH 7.4 and 5.0. The scale bars are 100 nmin the TEM images.

FIG. 33 shows the fluorescence spectra of FRET polymer pairs ofPEO-b-P(DPA-r-TMR)/PEO-b-P(DPA-r-Cy5) at different initial concentrationof Cl⁻ (0-2,000 mM). Different concentrations of ClO₄ ⁻ (in M) anionswere added to induce micelle formation. The samples were excited atλ_(ex)=545 nm and emission spectra were collected from 560-750 nm. Allthe experiments were conducted at pH=4, below the transition pH ofPEO-b-PDPA (pH_(t)=6.1).

FIG. 34 shows the FRET transfer efficiency as a function of ClO₄ ⁻concentration at different competing Cl⁻ concentrations (0-2,000 mM).

FIG. 35 shows the fluorescence spectra of FRET polymer pairs ofPEO-b-P(DPA-r-TMR)/PEO-b-P(DPA-r-Cy5) at different initial concentrationof SO₄ ²⁻ (0-500 mM). Different concentrations of ClO₄ ⁻ (in M) anionswere added to induce micelle formation. The samples were excited atλ_(ex)=545 nm and emission spectra were collected from 560-750 nm. Allthe experiments were conducted at pH=4, below the transition pH ofPEO-b-PDPA (pH_(t)=6.1).

FIG. 36 shows the FRET transfer efficiency as a function of ClO₄ ⁻concentration at different competing SO₄ ²⁻ concentrations (0-500 mM).

FIGS. 37A & B show (FIG. 37A) ClO₄ ⁻-induced self-assembly of copolymer3 in the presence of different concentrations of competing SO₄ ²⁻anions. (FIG. 37B) The FRET efficiency (FC₅₀) from ClO₄ ⁻-inducedself-assembly as a function of ionic strength of competing Cl⁻ and SO₄²⁻ anions. The solution pH was controlled at 4 in these studies.

FIGS. 38A & B show (FIG. 38A) hydrophobic strength of PR segment affectsthe ability of ClO₄ ⁻ in micelle induction. More hydrophobic PR segment(e.g., pentyl groups in 5) increases the ClO₄ ⁻ sensitivity to inducemicelle formation. (FIG. 38B) An empirical model depicting two importantcontributing factors (hydrophobic alkyl chain length and chaotropicanions) on the self-assembly of ionic polymeric micelles.

FIG. 39 shows the fluorescence spectra of FRET polymer pairs ofPEO-b-P(R-r-TMR)/PEO-b-P(R-r-Cy5) of different hydrophobic strengths (PRsegments were varied from methyl to pentyl side chains). Differentconcentrations of ClO₄ ⁻ (in M) anions were added to induce micelleformation. The samples were excited at λ_(ex)=545 nm and emissionspectra were collected from 560-750 nm. All the experiments wereconducted at pH=4, which was below the transition pH of PEO-b-PDPA(pH_(t)=6.1).

FIGS. 40A-C show the UPS_(6.9) nanoprobe with exquisitely sharp pHtransition at 6.9. (FIG. 40A) Structure of the ionizable block copolymerand its pH-dependent fluorescence emission properties. At high pH (i.e.,7.4 or 7.2), UPS stays silent. At pH below 6.9, UPS is activated as aresult of micelle dissociation. The pH response is much sharper than ahypothetical small molecular pH sensor (blue dashed line). (FIG. 40B)Fluorescent images of UPS_(6.9) solution in different pH buffers. (FIG.40C) Transmission electron micrographs of UPS_(6.9) in the micelle andunimer states at pH 7.4 and 6.7, respectively (polymer concentration=1mg/mL, scale bar=100 nm).

FIGS. 41A & 41B show the PK/BD of two UPS nanoprobes with comparablesize but different pH transitions (pH_(t)=6.3 and 6.9).

FIGS. 42A & 42B show (FIG. 42A) NIR image of a representative sentinellymph node on the side of the neck after removal of primary HNSCCtumors. (FIG. 42B) Histology (H&E) was able to validate the nodalstructures. The selected node showed presence of HN5 cells (blackarrows) in the cortex region of the node.

FIGS. 43A-43I show the syntheses and optimization of PINS nanoprobes.FIG. 43A Schematic syntheses of ICG-conjugatedPEG-b-P(EPA_(x)-r-ICG_(y)) block copolymers. FIGS. 43B-43D Investigationof the influence of the PEPA segment length on the pH-dependentfluorescence properties: (FIG. 43B) fluorescence intensity, (FIG. 43C)fluorescence activation ratio at pH of interest over 7.4, and (FIG. 43D)normalized fluorescence intensity. The PEPA segment length was varied(x=40, 60, 80, 100, 120) while the number of ICG per polymer chain wasmaintained at 1. FIGS. 43E-G: Investigation of the influence of ICGconjugation number on the pH-dependent fluorescence properties: (FIG.43E) fluorescence intensity, (FIG. 43F) fluorescence activation ratio atpH of interest over 7.4, and (FIG. 43G) normalized fluorescenceintensity. The number of ICG per polymer chain was varied (y=0.5, 1, 2)while the PEPA segment length was controlled at 100. FIGS. 43H & 43I:UV-Vis absorption spectra with normalization to the monomer peakintensity (λ=808 nm) of PEPA₁₀₀-ICG_(y)(n=0.5, 1, 2) in (FIG. 43H) humanserum at pH 7.4 and (FIG. 43I) human serum at pH 6.0. Based on thesedata, PEG-b-P(EPA₁₀₀-r-ICG₁) was chosen as the optimal composition foranimal imaging studies.

FIGS. 44A-44F show characterization of PINS. FIG. 44A A 3D plot offluorescence intensity as a function of PINS concentration and pH. FIG.44B Near IR images of PINS solution by SPY Elite® surgical camerashowing pH-sensitive off/on activation. FIG. 44C Transmission electronmicrographs of PINS in the micelle and unimer states at pH 7.4 and 6.5,respectively. Polymer concentration=1 mg/mL; scale bars=100 nm. PINSfluorescence intensity at pH 6.5 (black bars) and 7.4 (white bars) inPBS (FIG. 44D) or 50% human serum (FIG. 44E) upon storage. FIG. 44FNumber-weighted hydrodynamic radius of PINS nanoprobes upon storage.Storage condition for (FIG. 44D)-(FIG. 44F): 10% w/v sucrose solution at−20° C. These results show PINS was stable in storage over 6 months in10% w/v sucrose solution at −20° C.

FIGS. 45A-45E show the dose-response of PINS in mice bearing human HN5orthotopic tumors. White light (FIG. 45A) and near IR (FIG. 45B) imagesof mice injected with different doses of PINS (1.0, 2.5 and 5.0 mg/kg)via the tail veins. HN5 tumor intensity increased with increasing PINSdose. Free ICG control at an equivalent dye dose to 2.5 mg/kg PINS didnot show observable tumor contrast. FIG. 45C: NIR images ofrepresentative mice injected with different doses of PINS at selectedtime points. Quantification of tumor fluorescence intensity (FIG. 45D)and tumor contrast over noise ratio (FIG. 45E) as a function of timeafter intravenous injection (n=3). Higher PINS dose at 5.0 mg/kg led toreduced CNR value due to the higher background signal in muscle tissue.Based on results from FIG. 45E, 2.5 mg/kg was chosen as the optimal PINSdose for tumor acidosis imaging.

FIGS. 46A & 46B show tumor acidosis imaging by PINS. FIG. 46A Schematicof tumor metabolic imaging by PET with FDG or NIR fluorescence imagingwith PINS. FIG. 46B Comparison of FDG-PET with PINS imaging in SCID micebearing large or small HN5 orthotopic tumors. PINS imaging showeddramatically improved sensitivity and specificity of tumor detectionover FDG-PET. Additional comparisons are available in FIGS. 47A-47E (n=3for each animal group).

FIGS. 47A-47E show the comparison of FDG-PET with PINS imaging in micebearing orthotopic HN5 tumors. White light, FDG-PET/CT and NIR imagesfor the same group of mice with large tumors (200 mm³, FIG. 47A) orsmall tumors (15 mm³, FIG. 47B). PINS imaging allowed clear tumor margindelineation for all big and small tumors. For big tumors, FDG-PET showedhigher signal on the periphery of the tumors consist with PINSactivation. Sagittal view of the same group of mice with large (FIG.47C) and small tumors (FIG. 47D). FIG. 47E Stitched H&E images for thebig and small tumors shown in FIGS. 46A & 46B. Scale bars: 2 mm in bigtumor and 500 am in small tumor images.

FIG. 48 shows PINS imaging achieved broad tumor specificity. PINSnanoprobes (2.5 mg/kg, i.v. injection 24 h prior to imaging by SPYElite® clinical camera) demonstrate broad tumor imaging efficacy indifferent tumor models (head and neck, breast, peritoneal mets, kidney,brain) and organ sites. Arrow heads indicate the location of tumors.

FIGS. 49A & 49B show the compatibility of PINS nanoprobes with differentclinical cameras. FIG. 49A: Clinically used ICG imaging systems: NovadaqSPY Elite®, Hamamastu PDE and Leica FL-800 models. FIG. 49B: White lightand NIR images of the same tumor bearing mouse under different clinicalICG imaging systems.

FIGS. 50A-50F show ex vivo organ and tumor fluorescence imaging afterPINS injection. NIR images of main organs and quantification of organ tomuscle ratios of fluorescence intensity 24 h after injection ofnanoprobes in mice bearing (FIG. 50A) HN5, (FIG. 50B) FaDu and (FIG.50C) HCC4034 head and neck tumors, (FIG. 50D) MBA-MD-231 and (FIG. 50E)4T1 breast tumors, and (FIG. 50F) U87 glioma. Data are presented asmean±s.d. (n=3). Livers were not calculated due to signal saturation.

FIGS. 51A & 51B show tumor acidosis guided surgery (TAGS) in micebearing orthotopic head and neck tumors. FIG. 51A Surgical resection ofprimary HN5 tumors and successful detection of residual tumors by SPYElite® camera. Visual inspection of tumor bed by eyes was not able todifferentiate residual tumors from surrounding muscle tissue (top left).Tumor tissue (T) and normal tissue (N) were verified by histology. Scalebar=1 mm (low magnification) or 100 μm (high magnification). FIG. 51B Asexpected debulking surgery provided no survival benefit over untreatedcontrol. TAGS shows significantly improved long-term survival over whitelight and other control groups (****P<0.0001). For control and debulkinggroup n=7; for white light group n=15; for TAGS group n=18.

FIG. 52 shows histology validation of primary tumor, tumor margin andnegative bed. Five representative H&E histology images from each type ofspecimens collected during the non-survival surgeries. Arrow headsindicate the presence of cancer cells in the tumor margin specimens.Scale bar=1 mm (top rows, low magnification) or 100 μm (bottom rows,high magnification).

FIGS. 53A-53D show tumor acidosis guided surgery in mice bearing smalloccult breast tumor nodules. Tumor foci (<1 million cells) was visibleunder SPY camera (FIG. 53B) but not by visual detection (FIG. 53A). FIG.53C: A representative histology section of a small breast tumor noduleresected during TAGS; scale bar=200 μm. FIG. 53D: Kaplan-Meier curvedemonstrates significantly improved long-term survival by TAGS overwhite light and untreated control groups. For control groups n=7; whitelight and TAGS groups n=10; *P<0.05.

FIGS. 54A-54C show the evaluation of small molecular inhibitorstargeting different tumor acidosis pathways by PINS. FIG. 54A: Chemicalstructure of selected small molecular inhibitors and their correspondingtargets in parenthesis: acetazolamide (CAIX), α-cyano-4-hydroxycinnamateor CHC (MCT), cariporide (NHE1) and pantoprazole (proton pump). FIG.54B: Representative images of mice bearing triple negative 4T1 breasttumors in immunocompetent BalB/C mice after injection of PBS or othertumor acidosis inhibitors. FIG. 54C: Quantification of NIR fluorescenceimages shown in FIG. 54B. The fluorescence intensity was normalized tothe PBS control. CAIX inhibition by acetazolamide resulted in the mostefficient suppression of tumor acidosis.

FIGS. 55A-55E show the safety assessment of intravenously administeredPINS in healthy C57BL/6 mice. FIG. 55A: Normalized change of body weightof C57BL/6 immunocompetent mice after bolus injection of 200 or 250mg/kg PINS compared to PBS control. FIGS. 55B-55E: Serum tests for liver(FIGS. 55B & 55C) and kidney (FIGS. 55D & 55E) functions of C57BL/6immunocompetent mice after bolus injection of PINS at different dosesand sacrificed after selected time points. For all groups n=5.Abbreviations: ALT, alanine aminotransferase; GOT, glutamic oxaloacetictransaminase; BUN, blood urea nitrogen; CRE, creatinine; dotted linesindicate typical wild-type mean values for C57BL/6 mice.

FIG. 56 shows the histology analyses of major organs for safetyassessment of PINS. Representative H&E sections of the main organs fromC57BL/6 immunocompetent mice after bolus injection (250 mg/kg) orrepeated injection (25 mg/kg/week, 5 injections) of PINS and sacrificedafter selected time points (n=5 for each group). At 250 mg/kg,microsteatosis was observed in the liver at earlier time points (day 1and day 7), but recovered on day 28. Spleen, kidney and heart showed noabnormalities. For repeated injection, no abnormalities were observed inany of the main organs.

FIGS. 57A-57C show a UPS nanoparticle library with sharply definedbuffer capacity across a broad physiological pH range. (FIG. 57A)Schematic illustration of the buffer effect of UPS nanoparticles and thechemical structures of PEO-b-P(R₁-r-R₂) copolymers with finely tunablehydrophobicity and pK_(a). The composition for each copolymer is shownin Table 11. (FIG. 57B) pH titration of solutions containing UPS_(6.2),UPS_(5.3) and UPS_(4.4) nanoparticles using 0.4 M HCl. The maximumbuffer pH corresponds to the apparent pK_(a) of each copolymer.Chloroquine (CQ, pK_(a)=8.3 and 10.4), a small molecular base, andpolyethyleneimines (PEI) were included for comparison. (FIG. 57C) Buffercapacity (β) for each component of the UPS library was plotted as afunction of pH in the pH range of 4.0 to 7.4. At different pH values,UPS nanoparticles were 30-300 fold higher in buffer strength over CQ.L.E. and E.E. are abbreviations for late endosomes and early endosomes,respectively.

FIG. 58 shows the syntheses of dye-conjugated diblock copolymers. The PRsegment consists of a random block from two monomers with differentmolar fractions to fine-tune its hydrophobicity and pH transition (seeTable 10). The structure of Cy5 dye is also shown.

FIG. 59 shows the TEM images of the UPS nanoprobe library. Nanoprobeswere dissolved in PBS buffer (pH 7.4) and dried on a carbon grid priorto TEM analysis. Phosphotungstic acid was used for negative staining.Scale bar=100 nm for all images.

FIG. 60 shows the pH titration of each component of the UPS nanoprobelibrary. HCl (0.4 M) was added incrementally to titrate micelle solution(2 mg/mL polymer concentration or 8 mM based on the amount of aminegroups) of all ten UPS nanoprobes, choloroquine solution (2 mg/mL or12.5 mM based on the amount of amine groups) and PEI (branched, MW10,000 Da, Polyscience, Inc.) solution (0.3 mg/mL or 7.3 mM based on theamount of amine groups). A pH/conductivity meter (Mettler Toledo) wasused to monitor the change of pH in the solution during titration.

FIGS. 61A-61C show the syntheses and characterization ofAlways-ON/OFF-ON UPS nanoparticles. (FIG. 61A) and (FIG. 61B) Schematicof the dual-reporter nanoparticle. In the micelle state, the Always-ONdyes serve as the quencher for the ON/OFF fluorophores. When the micelleis disassembled, the Always-ON and ON/OFF fluorophores can fluoresceindependently. The ON/OFF ratio of BODIPY (FIG. 61A) and Cy3.5 (FIG.61B) varies when the ratio of polymers conjugated with these two dyesvaries. Weight fraction of 60% BODIPY-conjugated copolymer with 40%Cy3.5-conjugated copolymer was chosen as the final combination. (FIG.61C) Fluorescence signal amplification of UPS_(6.2) nanoprobes as afunction of pH. Images were captured on Maestro in vivo imaging system(CRI) using the green and yellow filters.

FIGS. 62A-62C show the pH-sensitive imaging and buffering of endocyticorganelles in HeLa cells. (FIG. 62A) Representative confocal images ofHeLa cells at the indicated time points following a 5 min exposure tolow (100 μg/mL) and high dose (1,000 μg/mL) of UPS_(6.2). Nuclei werestained blue with Hoechst. Scale bar=10 μm. (FIG. 62B) Quantitativeanalysis of the activation kinetics of always-ON/OFF-ON UPS_(6.2.) Thefluorescent intensity of punctae in BODIPY channel (OFF-ON) wasnormalized to that of Cy3.5 (always-ON). (FIG. 62C) Real-timemeasurement of endo/lysosomal pH in HeLa cells treated with theindicated doses of UPS_(6.2). Lysosensor ratiometric imaging probe wasused for in situ pH measurement. The error bars represent standarddeviations from 50 organelles at each time point.

FIGS. 63A-63C show the Buffering endocytic organelles of HeLa cells withUPS_(5.3) nanoprobes. (FIG. 63A) Confocal images of HeLa cells at theindicated time points following a 5 min exposure to low dose (100 μg/mL)and high dose (1,000 μg/mL) Always-ON (Cy3.5)/OFF-ON (BODIPY) UPS_(5.3).Nuclei were stained blue with Hoechst. Scale bar=10 μm. (FIG. 63B)Quantitative analysis of the OFF-ON activation process of UPS_(5.3). Thefluorescent intensity of punctae in cells in BODIPY channel wasnormalized to the fluorescent intensity of the same puncta in TMRchannel. (FIG. 63C) Real-time measurement of endo/lysosomal pHfluctuation in HeLa cells treated with always-ON (Cy3.5)/OFF-ON (BODIPY)UPS_(5.3) at indicated doses. Lysosensor Yellow/Blue DND160 ratiometricimaging probe was used for in situ pH measurement. The error barsrepresent standard deviations from 50 organelles at each time point.

FIGS. 64A-64F show the buffering the pH of endocytic organelles affectstheir membrane protein dynamics. HeLa cells were treated with 1,000μg/mL UPS_(6.2)-Cy5 or UPS_(4.4)-Cy5 for 5 min for cell uptake. Thenthey were incubated for 15 min (FIG. 64A), 1 h (FIG. 64B) and 2 h (FIG.64C) before fixation. Immunofluorescence (IF) images show thelocalization of UPS nanoprobes in early endosomes (Rab5) or lysosomes(LAMP2). Scale bar=10 μm and 5 μm (inset). Imaris software was used toanalyze colocalization of z-stacked confocal images. The faction of UPScolocalized with Rab5 (FIG. 64D) and LAMP2 (FIG. 64E) and the faction ofRab5 colocalized with LAMP2 (FIG. 64F) were calculated from Mander'scoefficient, n=10, α=0.05, ****p<0.0001. Two-way ANOVA and Sidak'smultiple comparison tests were performed to assess the statisticalsignificance.

FIGS. 65A-65E show the clamping luminal pH of endo-lysosomes with UPSselectively inhibits amino acid-dependent mTORC1 activation. HeLa cellswere starved in EBSS for 2 h and then stimulated with essential aminoacids (EAAs) for indicated time intervals in the presence of (FIG. 65A)UPS_(6.2)/UPS_(5.3)/UPS_(5.0) and (FIG. 65B) UPS_(4.7)/UPS_(4.4). Waterand 50 μM chloroquine (CQ) were used as control. Accumulation of theindicated phosphoproteins was assessed by immunoblot of whole celllysates. (FIG. 65C) Quantitative analysis of the nuclear/cystosolicdistribution of GFP-TFEB following the indicated treatments. Error barsrepresent standard deviation, n=10. (FIG. 65D) Representative images forFIG. 65C. Scale bar=10 μm. (FIG. 65E) Working model of pH transitionsrequired for free amino acid versus albumin-derived amino acid dependentactivation of the mTORC1 signaling pathway.

FIGS. 66A-66C show the mTORC1 signal quantitation and cathepsin Bactivity upon UPS exposure. (FIGS. 66A & 66B) Quantitative analysis ofphosphorylated S6 protein normalized by its total protein levels inFIGS. 64A & 64B. Statistic difference between control (water) and UPStreated groups at each time point was detected by two-way ANOVA andDunnett's multiple comparison test, α=0.05, **p<0.01, ****p<0.0001 ornot significant (n.s.). The statistical differences between control andUPS treated groups were not significant at any time point in FIG. 66B.Error bars indicate standard deviation, n=3. (FIG. 66C) Cathepsin Bactivity was measured in response to the indicated treatments (n=2).Statistical difference between ‘Fed’ and all the other groups wasdetected by one-way ANOVA and Dunnett's multiple comparison test,α=0.05, *p<0.05 or not significant (n.s.).

FIGS. 67A-67D show the albumin-dependent mTORC1 pathway activation isinhibited by UPS_(4.4). (FIG. 67A) HeLa cells were deprived of nutrientsfor 2 h followed by BSA uptake (2%) in the presence or absence of theindicated UPS nanoparticles (1,000 μg/ml). Accumulation of the indicatedphosphoproteins was monitored by immunoblot of whole cell lysates. (FIG.67B) Nuclear/cytosolic distribution of GFP-tagged TFEB was monitored inresponse to the indicated conditions. (FIG. 67C) Quantitative analysisof phosphorylated S6 protein normalized by its total protein levels in(FIG. 67A). Error bars indicate standard deviation, n=3. Statisticdifference between control and UPS treated groups at each time point wasdetected by two-way ANOVA and Dunnett's multiple comparison test,α=0.05, **p<0.01, ****p<0.0001 or not significant (n.s.). (FIG. 67D)Quantitative analysis of the location of TFEB in the results shown in(FIG. 67B) (in the cytosol=0, in the nucleus=1). The error barsrepresent standard deviation. Scale bar=10 μm.

FIGS. 68A-68C show the selective buffering of lysosomal pH modulates thecellular metabolite pool. (FIG. 68A) Dendrogram indicates relativeabundance of the indicated metabolites in nutrient replete (fed) ordeprived (starved) medium as normalized to the total protein content.Cells were treated with UPS_(4.4) at the indicated doses. (FIG. 68B)Normalized abundance of the selected amino acids under nutrient repleteand nutrient deprived conditions. Error bars represent standarddeviation, n=6. (FIG. 68C) A schematic indicating the consequence ofenvironment and lysosomal pH on the balance of cellular metabolitepools.

FIGS. 69A-69G show the UPS nanoparticles selectively kill NSCLC cellsthat are sensitive to lysosomal stress. (FIG. 69A) Schematic of the cellmodels employed and their corresponding vulnerabilities to lysosomalmaturation. (FIG. 69B) DIC images indicating the relative viability ofHBEC30 KT and HCC4017 cells with and without exposure to UPS ateffective doses (UPS_(6.2) and UPS_(5.3)=400 μg/ml, UPS_(4.4)=1,000g/ml). Scale bar=100 μm. (FIGS. 69C-69E) Caspase3/7 activity inHBEC30KT, HBEC30KT KP, HBEC30KT KPL and HCC4017 cells was measured 72 hafter exposure to the indicated doses of UPS. Two-way ANOVA and Sidak'smultiple comparison tests were performed to assess statisticalsignificance of observed differences between HBEC30KT and HCC4017, andHBEC30KT KP and HBEC30KT KPL, α=0.05, **p<0.01, ****p<0.0001. (FIGS. 69F& 69G) Cellular ATP levels were measured after exposure of HCC4017 (FIG.69F) and HBEC30 KT KPL (FIG. 69G) to 1,000 μg/mL UPS_(6.2) for 72 htogether with the indicated concentrations of methyl pyruvate (MP),dimethyl-2-oxoglutarate (MOG), or water (dash line). Values werenormalized to no treatment (without UPS) controls. Error bars indicatestandard deviation, n=4.

FIGS. 70A-70D show I-UPS_(6.9) nanoprobes. (FIG. 70A) Schematicsyntheses of ICG-conjugated block copolymers, PEG-b-P(C7A-r-ICG). (FIG.70B) Near IR images of I-UPS_(6.9) solution by SPY Elite® surgicalcamera showing pH-sensitive off/on activation. (FIG. 70C) Normalizedfluorescence intensity as a function of pH shows longer PC7A segmentleads to slightly lower pH transition and sharper response. (FIG. 70D) A3D plot of fluorescence intensity as a function of probe concentrationand pH. Data from FIGS. 70B-70D were obtained in 20% serum-containingsolutions.

FIG. 71 shows the proposed synthetic route of chelator conjugatedpolymer, CB-TE2A-PEG-PC7A_(x).

FIG. 72 shows mice bearing orthotopic HN5 tumors imaged by FDG-PET andI-UPS. Mice were imaged at sagittal position to show false positivesfrom brown fat or neck muscles (arrows) in FDG-PET images.

FIG. 73 shows tumor acidosis imaging by I-UPS with broad cancerspecificity in diverse tumor models (head and neck, breast, colorectalperitoneal mets, kidney, brain). Yellow arrow heads indicate thelocation of tumors. I—UPS_(6.9) (2.5 mg/kg) was i.v. injected 24 hbefore imaging by a SPY camera.

FIGS. 74A & 74B show (FIG. 74A) I-UPS_(6.9)-guided resection oforthotopic 4T1 breast tumors. Tumor foci (<1 million cells) was visibleunder SPY camera (right panel) but not by visual detection (left panel).(FIG. 74B) Kaplan-Meier curve demonstrates improved long-term survivalby I—UPS_(6.9)-guided resection over white light (P<0.05) and untreatedcontrol groups (P<0.01).

FIG. 75 shows UPS nanoprobe with Always-ON/OFF-ON dual reporter signals.UPS_(6.9) is used as an example and BODIPY/Cy3.5 as FRET donor/acceptorpair.

FIGS. 76A-76C show UPS nanoprobes with fine-tuned pH transitions. (FIG.76A) Synthetic scheme of PEG-b-PR copolymers with varying molarfractions of DPA-MA and DEA-MA subunits in the PR block. (FIG. 76B) pHresponse of the UPS nanoprobes for different PR compositions. Cy5.5 wasused as a model dye. (FIG. 76C) Transition pH as a function of molarpercentage of DPA establishes a standard curve for rational design ofUPS with pre-determined pH transition.

FIG. 77 shows pH regulatory machinery in a cancer cell. Proton pumpingresults in tumor acidosis in the microenvironment as well as raising theintracellular pH to promote cell proliferation and migration (Neri &Supuran, 2011).

FIGS. 78A-78E show UPS_(6.9) nanoprobes can specifically image tumorpH_(e) in A549 lung tumors. (FIG. 78A) Aerobic glycolysis convertsglucose to lactate in cancer cells. 2-DG and CHC are metabolicinhibitors for glucose uptake and lactic acid secretion, respectively.(FIG. 78B) Effect of 2-DG or CHC on the rate of lactic acid secretion inA549 cells. (FIG. 78C) Acidification of A549 cell culture medium in thepresence of 2-DG or CHC after 6 h incubation. *P<0.05, **P<0.01,***P<0.001, compared with vehicle group. (FIG. 78D) Overlaid fluorescentimages of A549 tumor-bearing mice at 24 h post-injection of UPS_(6.9)(10 mg/kg). In the control groups, 2-DG (250 mg/kg) or CHC (250 mg/kg)was injected 12 h before UPS_(6.9) administration. Cy5.5 (light spot)and autofluorescence (light background) are shown in the compositeimages. (FIG. 78E) NIR fluorescence intensity ratio between tumor andnormal tissues (T/N ratio) as a function of time after UPS_(6.9)injection. Data are presented as mean±s.d. (n=4).

FIGS. 79A-79C show I-UPS imaging of orthotopic 4T1 breast tumors. Tumorfoci (<1 million cells) was visible under SPY camera (heat mode, FIG.79B) but not by visual detection (FIG. 79A). Tumor presence was verifiedby histology (FIG. 79C). The scale bar is 200 μm in FIG. 79C.

FIG. 80 shows mice bearing orthotopic HN5 tumors imaged by FDG-PET andI-UPS. Mice were imaged at sagittal position to show false positivesfrom brown adipose tissue in the neck (2 out 3 mice) in FDG-PET images.

FIG. 81 shows the rates of glucose consumption and lactate secretionfrom a panel of 80 human non-small cell lung cancer cells. These lungcancer cells display a divergent glycolysis rates as represented by theratio of Lac_(out)/Glu_(in).

FIG. 82 shows schematic quantifying the BODIPY and Cy3.5 signals andcorrelating with true tumor margin delineated by histology. Fluorescenceintensity along the perpendicular line to a tumor tangent point will bemeasured. Averaged intensity vs distance will be determined frommultiple tangent points along tumor margins.

FIGS. 83A-B show comparison of PK/BD of two UPS nanoprobes withcomparable size but different pH transitions.

FIGS. 84A-84C show the schematic design and working principle of themulti-spectral hybrid UPS nanoprobe. FIG. 84A: The multi-spectral hybridUPS nanoprobe is engineered by three PEG-b-PR block copolymers eachencoded with different fluorescent dyes. The hybrid UPS nanoprobe stays“OFF” at neutral pH. When the pH is lowered, the PEG-b-(PR-r-dye)components disassemble and fluoresce sequentially to present differentcolors upon encounting subtle pH changes. FIG. 84B: The chemicalstructures of the PEG-b-PR block copolymers and fluorescent dyeconjugated polymers. FIG. 84C: The internalization and activation of themulti-spectral hybrid UPS nanoprobe in live cells through thereceptor-mediated endocytosis, such as endothelial growth factorreceptor (EGFR). After internalization, the PEPA-BDY493 is turned ON byclathrin-coated vesicles (CCV, pH˜6.8), then the PDPA-TMR is activatedby early endosomes (pH˜6.0), finally the PDBA-Cy5 is turned ON by thelate endosome/lysosome (pH˜5.0-5.5).

FIGS. 85A & 85B show (FIG. 85A) pH-dependent fluorescence emissionspectra and (FIG. 85B) fluorescence intensity ratio of PEPA-BDY493 as afunction of pH in 0.1 M PBS solution. The samples are excited at 488 nm,and the emission spectra are collected from 500 to 650 nm. The polymerconcentrations are controlled at 0.1 mg/mL.

FIGS. 86A & 86B show (FIG. 86A) pH-dependent fluorescence emissionspectra and (FIG. 86B) fluorescence intensity ratio of PDPA-TMR as afunction of pH in 0.1 M PBS solution. The samples are excited at 545 nm,and the emission spectra are collected from 560 to 750 nm. The polymerconcentrations are controlled at 0.1 mg/mL.

FIGS. 87A & 87B show (FIG. 87A) pH-dependent fluorescence emissionspectra and (FIG. 87B) fluorescence intensity ratio of PDBA-Cy5 as afunction of pH in 0.1 M PBS solution. The samples are excited at 640 nm,and the emission spectra are collected from 650 to 750 nm. The polymerconcentrations are controlled at 0.1 mg/mL.

FIG. 88 shows normalized fluorescence intensity as a function of pH forPEPA-BDY493, PDPA-TMR, and PDBA-Cy5 micelles. The polymer concentrationswere 100 g/mL.

FIGS. 89A & 89B show fluorescence characterization of molecularly mixedmicelles and micelle mixture. (FIG. 89A) Fluorescence intensity ofmolecularly mixed micelles of PDBA-Cy5 and PEG-b-(PR-r-AMA₃) with molarratio of 1:19 at pH 7.4. (FIG. 89B) Fluorescence intensity of micellesmixture of PDBA-Cy5 micelle and PEG-b-(PR-r-AMA₃) micelle with molarratio of 1:19 at pH 7.4.

FIGS. 90A-90D show fluorescent resonance energy transfer (FRET)experiments demonstrate the formation of the multi-spectral hybrid UPSnanoprobe. The PEG-b-PR block copolymers are encoded with differentdyes. Three exemplary PEG-b-(PR-r-Dye) block copolymers, includingPEPA-BDY493, PDPA-TMR, and PDBA-Cy5 are synthesized with a lowdye/polymer ratio (1:1) to minimize the homoFRET effect. FIG. 90A: ThePEPA-BDY493, PDPA-TMR, PEPA-BDY493/PDPA-TMR (1:1) micelles, and micellemixture of PEPA-BDY493 and PDPA-TMR are excited at 485 nm, then theemission spectra are collected from 490 to 720 nm. A strong FRET effectfrom PEPA-BDY493 to PDPA-TMR is observed, indicating the formation ofthe PEPA/PDPA hybrid nanoparticle. FIG. 90B: A strong FRET effect fromPDPA-TMR to PDBA-Cy5 is observed, indicating the formation of thePDPA/PDBA hybrid nanoparticle. FIG. 90C: A strong FRET effect fromPEPA-BDY493 to PDBA-Cy5 is observed, indicating the formation of thePEPA/PDBA hybrid nanoparticle. FIG. 90D: A strong sequential FRETeffects from BDY493 to TMR, finally to Cy5 are observed, indicating theformation of the PEPA/PDPA/PDBA hybrid nanoparticle.

FIG. 91 shows representative images of dye-conjugated polymericmicelles, including PEPA-BDY493 (1), PDPA-TMR (2), PDBA-Cy5 (3), andthree-in-one hybrid nanoprobe (4).

FIGS. 92A-92F show the in vitro characterization of the hybridnanoprobe. (FIGS. 92A-92D) Fluorescence spectra of the hybrid nanoprobein different pH buffers. The BDY493, TMR, and Cy5 signals are excited at485, 545, and 640 nm, respectively. The emission spectra for BDY493,TMR, and Cy5 are collected from 490-750 nm, 560-750, and 650-750 nm,respectively. (FIG. 92E) The count rates and normalized fluorescenceintensity of hybrid nanoprobe as a function of time are plotted. Thecount rates at different pH are determined by dynamic light scatteringanalysis. The multi-stage activation of the multi-spectral hybrid UPSnanoprobe is shown by green, red, and blue sigmoidal curves at differentpH ranges. (FIG. 92F) Representative fluorescence images ofmulti-spectral UPS nanoprobe at different pH are captured. Yellow is themerged color of green and red signals. White is the merged color ofblue, green, and red signals.

FIGS. 93A-93F show pH-dependent fluorescence emission spectra of hybridnanoprobe. BDY493, TMR, and Cy5 signals were excited at 485, 545, and640 nm, respectively. The corresponding emission spectra were collectedat 490-750, 560-750, and 650-750 nm, respectively.

FIG. 94 shows normalized fluorescence intensity of PEPA-BDY493,PDPA-TMR, and PDBA-Cy5 components in hybrid UPS nanoprobe as a functionof pH.

FIGS. 95A & 95B show (FIG. 95A) TEM and (FIG. 95B) DLS analyses ofmorphology and particle size distribution of hybrid nanoprobes atdifferent pH solution. The scale bar is 100 nm in the TEM images.

FIG. 96 shows specific fluorescence activation of Erbitux-conjugatedPDPA-TMR nanoprobes in A549 cells. The cells were treated withErbitux-conjugated PDPA-TMR micelle (upper) or PDPA-TMR micelles (lower)for 1 hour, respectively. The scale bar is 40 am.

FIG. 97 shows synchronized uptake of Erbitux-encoded hybrid UPSnanoprobes in single endocytic organelle of A549 human lung cancercells. Tumor cells were incubated with nanoprobes for 3 hours followedby confocal imaging. The scale bar is 40 μm.

FIG. 98 shows multiplexed imaging of endosome maturation bymulti-colored hybrid nanoprobes in lung cancer A549 cells. Scale bar is20 μm.

FIG. 99 shows multiplexed imaging of endosome maturation bymulti-colored hybrid nanoprobes in head and neck cancer HN5 cells. Scalebar is 20 μm.

FIG. 100 shows multiplexed imaging of endosome maturation bymulti-colored hybrid nanoprobes in human lung cancer H460 cell line,which has KRAS gene mutation. Scale bar is 20 μm.

FIG. 101 shows multiplexed imaging of endosome maturation bymulti-colored hybrid nanoprobes in human lung cancer A549 cell line,which has KRAS gene mutation. Scale bar is 20 μm.

FIG. 102 shows multiplexed imaging of endosome maturation bymulti-colored hybrid nanoprobes in human lung cancer H2882 cell line,which has P53 gene mutation. Scale bar is 20 μm.

FIGS. 103A & 103B show organelle maturation in a panel of lung cancercell lines. Quantification of maturation rates of early endosomes (FIG.103A) and late endosomes/lysosomes (FIG. 103B) pinpoint Kras mutationbeing responsible for the phenotypic difference. The fluorescenceintensity of PDPA-TMR (I_(6.2)) in early endosome (EE) at 30 min isnormalized by PEPA-BDY493 signals (I_(6.9)). The fluorescence intensityof PDBA-Cy5 (I_(5.3)) in late endosome/lysosome (LE/Lys) at 75 min isnormalized by PEPA-BDY493 signals (I_(6.9)). Significant differencebetween Kras mutated cell lines and Kras wild type cell lines indicatesKRAS mutation is responsible for the late endosome/lysosome maturation.**P<0.01, paired, two-sided t-test; n=10.

FIGS. 104A-104D show organelle maturation in a panel of lung cancer celllines. Quantification of maturation rates of early endosomes (FIG. 104A)and late endosomes/lysosomes (FIG. 104B) pinpoint Kras mutation beingresponsible for the phenotypic difference. FIG. 104C: The fluorescenceintensity of PDPA-TMR (I_(6.2)) in early endosome (EE) at 30 min isnormalized by PEPA-BDY493 signals (I_(6.9)). FIG. 104D: The fluorescenceintensity of PDBA-Cy5 (I_(5.3)) in late endosome/lysosome (LE/Lys) at 75min is normalized by PEPA-BDY493 signals (I_(6.9)). Significantdifference between K-ras mutated cell lines and K-ras wild-type celllines indicates KRAS mutation is responsible for the lateendosome/lysosome maturation. **P<0.01, paired, two-sided t-test; n=10.

FIGS. 105A & 105B shows multistage pH imaging of organelle maturationduring endocytosis using multi-spectral hybrid UPS nanoprobes in livingcells. HBEC30 lung epithelial cells (FIG. 105A) and isogenic HCC4017lung cancer cells (FIG. 105B) are incubated with 100 ag/mLErbitux-conjugated hybrid UPS nanoprobe at 4° C. for 30 min, washed, andimaged in real time at 37° C. under a confocal microscope. ThePEPA-BDY493, PDPA-TMR, and PDBA-Cy5 signals are excited at 488, 543, and637 nm, respectively. FITC (515/30BP), TRITC (590/75BP), and Cy5 (650LP)filters are used for PEPA-BDY493, PDPA-TMR, and PDBA-Cy5 image capture,respectively. BDY493, TMR, and Cy5 are shown as green, red, and bluecolors, respectively. The scale bar is 20 μm.

FIG. 106 shows multiplexed imaging of endosome maturation bymulti-colored hybrid nanoprobes in HBEC30KT human epithelial cells. Thescale bar is 20 am.

FIG. 107 shows multiplexed imaging of endosome maturation bymulti-colored hybrid nanoprobes in human isogenic HCC4017 lung cancercells. The scale bar is 20 am.

FIG. 108 shows multiplexed imaging of endosome maturation bymulti-colored hybrid nanoprobes in HBEC30KT-shTP53 cells. The scale baris 20 am.

FIG. 109 shows multiplexed imaging of endosome maturation bymulti-colored hybrid nanoprobes in HBEC30KT-shTP53/KRAS^(G12V) cells.The scale bar is 20 am.

FIG. 110 shows multiplexed imaging of endosome maturation bymulti-colored hybrid nanoprobes in HBEC30KT-shTP53/KRAS^(G12V)/shLKB1cells. The scale bar is 20 am.

FIGS. 111A-111D show time-course organelle maturation in an isogenicprogression series of HBEC30 cells. Quantification of maturation ratesof early endosomes (FIG. 111A) and late endosomes/lysosomes (FIG. 111B)pinpoint Kras mutation being responsible for the phenotypic difference.FIG. 111C: The fluorescence intensity of PDPA-TMR (I_(6.2)) in earlyendosome (EE) at 30 min was normalized by PEPA-BDY493 signals (I_(6.9)).Significant difference between HBEC30KT-shTP53 andHBEC30KT-shTP53/KRAS^(G12V) cells indicates KRAS mutation is responsiblefor the early endosome maturation. **P<0.01, paired, two-sided t-test;n=10. FIG. 111D: The fluorescence intensity of PDBA-Cy5 (I_(5.3)) inlate endosome/lysosome (LE/Lys) at 150 min was normalized by PEPA-BDY493signals (I_(6.9)). Significant difference between HBEC30KT-shTP53 andHBEC30KT-shTP53/KRAS^(G2)V cells indicates KRAS mutation is responsiblefor the late endosome/lysosome maturation. **P<0.01, paired, two-sidedt-test; n=10.

FIGS. 112A-112C show the syntheses and characterization ofAlways-ON/OFF-ON UPS nanoparticles. (FIG. 112A) and (FIG. 112B)Schematic of the dual-reporter nanoparticle. In the micelle state, theAlways-ON dyes serve as the quencher for the ON/OFF fluorophores. Whenthe micelle is disassembled, the Always-ON and ON/OFF fluorophores canfluoresce independently. The ON/OFF ratio of BODIPY (FIG. 112A) andCy3.5 (FIG. 112B) varies when the ratio of polymers conjugated withthese two dyes varies. Weight fraction of 60% BODIPY-conjugatedcopolymer with 40% Cy3.5-conjugated copolymer was chosen as the finalcombination. (FIG. 112C) Fluorescence signal amplification of UPS_(6.2)nanoprobes as a function of pH. Images were captured on Maestro in vivoimaging system (CRI) using the green and yellow filters.

FIGS. 113A & 113B show the chemical structures of triblock copolymersPEO-b-P(DEA-b-D5A), PEO-b-P(D5A₄₀-b-DEA₄₀) and random block polymerPEO-b-P(DEA-r-D5A) (FIG. 113A). pH titration curves forPEO-b-P(D5A₄₀-b-DEA₄₀), PEO-b-P(DEA₄₀-b-D5A₄₀) andPEO-b-P(D5A₄₀-r-DEA₄₀) copolymers as a function of molar fraction oftertiary amino groups (FIG. 113B).

FIGS. 114A & 114B show fluorescence characterization of molecularlymixed micelles and micelle mixture. (FIG. 114A) Fluorescence intensityof molecularly mixed micelles of PEPA-Cy5 and PEG-b-(PR-r-AMA₃) withmolar ratio of 1:19 at pH 7.4. (FIG. 114B) Fluorescence intensity ofmicelles mixture of PEPA-Cy5 micelle and PEG-b-(PR-r-AMA₃) micelle withmolar ratio of 1:19 at pH 7.4.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

In some aspects, the present disclosure provides a polymer which canform a pH responsive nanoparticle which dissembles above a particulartransition pH. In some embodiments, these polymers comprise a mixture ofdifferent monomers which allow specific tailoring of the desired pHtransition point (ΔpH₁₀₋₉₀%) of less than 0.25 pH units as well asdevelop pH probes for a range of pH transition points from about a pH of4 to about a pH of 8. The wide range of pH transition points allows fora wide range of application including but not limited to vesiculartrafficking, imaging of the pH_(e) of tumors, delivering drug compoundsto specific tissues, improving the visualization of a tumor to improvethe ability for a surgeon to resect the tumor tissue, or study thematuration or development of endosomes/lysosomes. In some aspects, thepresent disclosure provides methods of using these polymers in a pHresponsive system as described above. Additional methods of using thepolymers and the resultant pH responsive systems of the presentdisclosure are described in WO 2013/152059, which is incorporated hereinby reference.

A. CHEMICAL DEFINTIONS

When used in the context of a chemical group: “hydrogen” means —H;“hydroxy” means —OH; “carboxy” means —C(═O)OH (also written as —COOH or—CO₂H); “halo” means independently —F, —Cl, —Br or —I; “amino” means—NH₂; “nitro” means —NO₂; “cyano” means —CN; in a monovalent context“phosphate” means —OP(O)(OH)₂ or a deprotonated form thereof; in adivalent context “phosphate” means —OP(O)(OH)O- or a deprotonated formthereof; “mercapto” means —SH; and “thio” means ═S; “sulfonyl” means—S(O)₂—; and “sulfinyl” means —S(O)—.

In the context of chemical formulas, the symbol “—” means a single bond,“═” means a double bond, and “≡” means triple bond. The symbol “- - -”represents an optional bond, which if present is either single ordouble. The symbol

represents a bond or a double bond. Thus, for example, the formula

includes

And it is understood that no one such ring atom forms part of more thanone double bond. Furthermore, it is noted that the covalent bond symbol“—”, when connecting one or two stereogenic atoms, does not indicate anypreferred stereochemistry. Instead, it covers all stereoisomers as wellas mixtures thereof. The symbol “

”, when drawn perpendicularly across a bond (e.g.,

for methyl) indicates a point of attachment of the group. It is notedthat the point of attachment is typically only identified in this mannerfor larger groups in order to assist the reader in unambiguouslyidentifying a point of attachment. The symbol “

” means a single bond where the group attached to the thick end of thewedge is “out of the page.” The symbol “

” means a single bond where the attached to the thick end of the wedgeis “into the page”. The symbol “

” means a single bond where the geometry around a double bond (e.g.,either E or Z) is undefined. Both options, as well as combinationsthereof are therefore intended. Any undefined valency on an atom of astructure shown in this application implicitly represents a hydrogenatom bonded to that atom. A bold dot on a carbon atom indicates that thehydrogen attached to that carbon is oriented out of the plane of thepaper.

When a group “R” is depicted as a “floating group” on a ring system, forexample, in the formula:

then R may replace any hydrogen atom attached to any of the ring atoms,including a depicted, implied, or expressly defined hydrogen, so long asa stable structure is formed. When a group “R” is depicted as a“floating group” on a fused ring system, as for example in the formula:

then R may replace any hydrogen attached to any of the ring atoms ofeither of the fused rings unless specified otherwise. Replaceablehydrogens include depicted hydrogens (e.g., the hydrogen attached to thenitrogen in the formula above), implied hydrogens (e.g., a hydrogen ofthe formula above that is not shown but understood to be present),expressly defined hydrogens, and optional hydrogens whose presencedepends on the identity of a ring atom (e.g., a hydrogen attached togroup X, when X equals —CH—), so long as a stable structure is formed.In the example depicted, R may reside on either the 5-membered or the6-membered ring of the fused ring system. In the formula above, thesubscript letter “y” immediately following the group “R” enclosed inparentheses, represents a numeric variable. Unless specified otherwise,this variable can be 0, 1, 2, or any integer greater than 2, onlylimited by the maximum number of replaceable hydrogen atoms of the ringor ring system.

For the groups and classes below, the following parenthetical subscriptsfurther define the group/class as follows: “(Cn)” defines the exactnumber (n) of carbon atoms in the group/class. “(C≤n)” defines themaximum number (n) of carbon atoms that can be in the group/class, withthe minimum number as small as possible for the group in question, e.g.,it is understood that the minimum number of carbon atoms in the group“alkenyl_((C≤8))” or the class “alkene_((C<8))” is two. For example,“alkoxy_((C≤10))” designates those alkoxy groups having from 1 to 10carbon atoms. (Cn-n′) defines both the minimum (n) and maximum number(n′) of carbon atoms in the group. Similarly, “alkyl_((C2-10))”designates those alkyl groups having from 2 to 10 carbon atoms.

The term “saturated” as used herein means the compound or group somodified has no carbon-carbon double and no carbon-carbon triple bonds,except as noted below. In the case of substituted versions of saturatedgroups, one or more carbon oxygen double bond or a carbon nitrogendouble bond may be present. And when such a bond is present, thencarbon-carbon double bonds that may occur as part of keto-enoltautomerism or imine/enamine tautomerism are not precluded.

The term “aliphatic” when used without the “substituted” modifiersignifies that the compound/group so modified is an acyclic or cyclic,but non-aromatic hydrocarbon compound or group. In aliphaticcompounds/groups, the carbon atoms can be joined together in straightchains, branched chains, or non-aromatic rings (alicyclic). Aliphaticcompounds/groups can be saturated, that is joined by single bonds(alkanes/alkyl), or unsaturated, with one or more double bonds(alkenes/alkenyl) or with one or more triple bonds (alkynes/alkynyl).

The term “alkyl” when used without the “substituted” modifier refers toa monovalent saturated aliphatic group with a carbon atom as the pointof attachment, a linear or branched acyclic structure, and no atomsother than carbon and hydrogen. The groups —CH₃ (Me), —CH₂CH₃ (Et),—CH₂CH₂CH₃ (n-Pr or propyl), —CH(CH₃)₂ (i-Pr, iPr or isopropyl),—CH₂CH₂CH₂CH₃ (n-Bu), —CH(CH₃)CH₂CH₃ (sec-butyl), —CH₂CH(CH₃)₂(isobutyl), —C(CH₃)₃ (tert-butyl, t-butyl, t-Bu or ^(t)Bu), and—CH₂C(CH₃)₃ (neo-pentyl) are non-limiting examples of alkyl groups. Theterm “alkanediyl” when used without the “substituted” modifier refers toa divalent saturated aliphatic group, with one or two saturated carbonatom(s) as the point(s) of attachment, a linear or branched acyclicstructure, no carbon-carbon double or triple bonds, and no atoms otherthan carbon and hydrogen. The groups, —CH₂-(methylene), —CH₂CH₂—,—CH₂C(CH₃)₂CH₂-, and —CH₂CH₂CH₂—, are non-limiting examples ofalkanediyl groups. The term “alkylidene” when used without the“substituted” modifier refers to the divalent group ═CRR′ in which R andR′ are independently hydrogen or alkyl. Non-limiting examples ofalkylidene groups include: ═CH₂, ═CH(CH₂CH₃), and ═C(CH₃)₂. An “alkane”refers to the compound H-R, wherein R is alkyl as this term is definedabove. When any of these terms is used with the “substituted” modifierone or more hydrogen atom has been independently replaced by —OH, —F,—Cl, —Br, —I, —NH₂, —NO₂, —CO₂H, —CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃,—C(O)CH₃, —NHCH₃, —NHCH₂CH₃, —N(CH₃)₂, —C(O)NH₂, —OC(O)CH₃, or—S(O)₂NH₂. The following groups are non-limiting examples of substitutedalkyl groups: —CH₂OH, —CH₂Cl, —CF₃, —CH₂CN, —CH₂C(O)OH, —CH₂C(O)OCH₃,—CH₂C(O)NH₂, —CH₂C(O)CH₃, —CH₂OCH₃, —CH₂OC(O)CH₃, —CH₂NH₂, —CH₂N(CH₃)₂,and —CH₂CH₂Cl. The term “haloalkyl” is a subset of substituted alkyl, inwhich one or more hydrogen atoms has been substituted with a halo groupand no other atoms aside from carbon, hydrogen and halogen are present.The group, —CH₂Cl is a non-limiting example of a haloalkyl. The term“fluoroalkyl” is a subset of substituted alkyl, in which one or morehydrogen has been substituted with a fluoro group and no other atomsaside from carbon, hydrogen and fluorine are present. The groups, —CH₂F,—CF₃, and —CH₂CF₃ are non-limiting examples of fluoroalkyl groups.

The term “cycloalkyl” when used without the “substituted” modifierrefers to a monovalent saturated aliphatic group with a carbon atom asthe point of attachment, a linear or branched cyclo or cyclic structure,no carbon-carbon double or triple bonds, and no atoms other than carbonand hydrogen. As used herein, the cycloalkyl group may contain one ormore branching alkyl groups (carbon number limit permitting) attached tothe ring system so long as the point of attachment is the ring system.Non-limiting examples of cycloalkyl groups include: —CH(CH₂)₂(cyclopropyl), cyclobutyl, cyclopentyl, or cyclohexyl. The term“cycloalkanediyl” when used without the “substituted” modifier refers toa divalent saturated aliphatic group with one or two carbon atom as thepoint(s) of attachment, a linear or branched cyclo or cyclic structure,no carbon-carbon double or triple bonds, and no atoms other than carbonand hydrogen.

are non-limiting examples of cycloalkanediyl groups. The term“cycloalkylidene” when used without the “substituted” modifier refers tothe divalent group ═CRR′ in which R and R′ are taken together to form acycloalkanediyl group with at least two carbons. Non-limiting examplesof alkylidene groups include: ═C(CH₂)₂ and ═C(CH₂)₅. A “cycloalkane”refers to the compound H-R, wherein R is cycloalkyl as this term isdefined above. When any of these terms is used with the “substituted”modifier one or more hydrogen atom has been independently replaced by—OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H, —CO₂CH₃, —CN, —SH, —OCH₃,—OCH₂CH₃, —C(O)CH₃, —NHCH₃, —NHCH₂CH₃, —N(CH₃)₂, —C(O)NH₂, —OC(O)CH₃, or—S(O)₂NH₂. The following groups are non-limiting examples of substitutedcycloalkyl groups: —C(OH)(CH₂)₂, or.

The term “aryl” when used without the “substituted” modifier refers to amonovalent unsaturated aromatic group with an aromatic carbon atom asthe point of attachment, said carbon atom forming part of a one or moresix-membered aromatic ring structure, wherein the ring atoms are allcarbon, and wherein the group consists of no atoms other than carbon andhydrogen. If more than one ring is present, the rings may be fused orunfused. As used herein, the term does not preclude the presence of oneor more alkyl or aralkyl groups (carbon number limitation permitting)attached to the first aromatic ring or any additional aromatic ringpresent. Non-limiting examples of aryl groups include phenyl (Ph),methylphenyl, (dimethyl)phenyl, —C₆H₄CH₂CH₃ (ethylphenyl), naphthyl, anda monovalent group derived from biphenyl. The term “arenediyl” when usedwithout the “substituted” modifier refers to a divalent aromatic groupwith two aromatic carbon atoms as points of attachment, said carbonatoms forming part of one or more six-membered aromatic ringstructure(s) wherein the ring atoms are all carbon, and wherein themonovalent group consists of no atoms other than carbon and hydrogen. Asused herein, the term does not preclude the presence of one or morealkyl, aryl or aralkyl groups (carbon number limitation permitting)attached to the first aromatic ring or any additional aromatic ringpresent. If more than one ring is present, the rings may be fused orunfused. Unfused rings may be connected via one or more of thefollowing: a covalent bond, alkanediyl, or alkenediyl groups (carbonnumber limitation permitting). Non-limiting examples of arenediyl groupsinclude:

An “arene” refers to the compound H-R, wherein R is aryl as that term isdefined above. Benzene and toluene are non-limiting examples of arenes.When any of these terms are used with the “substituted” modifier one ormore hydrogen atom has been independently replaced by —OH, —F, —Cl, —Br,—I, —NH₂, —NO₂, —CO₂H, —CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃,—NHCH₃, —NHCH₂CH₃, —N(CH₃)₂, —C(O)NH₂, —OC(O)CH₃, or —S(O)₂NH₂.

The term “heteroaryl” when used without the “substituted” modifierrefers to a monovalent aromatic group with an aromatic carbon atom ornitrogen atom as the point of attachment, said carbon atom or nitrogenatom forming part of one or more aromatic ring structures wherein atleast one of the ring atoms is nitrogen, oxygen or sulfur, and whereinthe heteroaryl group consists of no atoms other than carbon, hydrogen,aromatic nitrogen, aromatic oxygen and aromatic sulfur. If more than onering is present, the rings may be fused or unfused. As used herein, theterm does not preclude the presence of one or more alkyl, aryl, and/oraralkyl groups (carbon number limitation permitting) attached to thearomatic ring or aromatic ring system. Non-limiting examples ofheteroaryl groups include furanyl, imidazolyl, indolyl, indazolyl (Im),isoxazolyl, methylpyridinyl, oxazolyl, phenylpyridinyl, pyridinyl,pyrrolyl, pyrimidinyl, pyrazinyl, quinolyl, quinazolyl, quinoxalinyl,triazinyl, tetrazolyl, thiazolyl, thienyl, and triazolyl. The term“N-heteroaryl” refers to a heteroaryl group with a nitrogen atom as thepoint of attachment. The term “heteroarenediyl” when used without the“substituted” modifier refers to an divalent aromatic group, with twoaromatic carbon atoms, two aromatic nitrogen atoms, or one aromaticcarbon atom and one aromatic nitrogen atom as the two points ofattachment, said atoms forming part of one or more aromatic ringstructure(s) wherein at least one of the ring atoms is nitrogen, oxygenor sulfur, and wherein the divalent group consists of no atoms otherthan carbon, hydrogen, aromatic nitrogen, aromatic oxygen and aromaticsulfur. If more than one ring is present, the rings may be fused orunfused. Unfused rings may be connected via one or more of thefollowing: a covalent bond, alkanediyl, or alkenediyl groups (carbonnumber limitation permitting). As used herein, the term does notpreclude the presence of one or more alkyl, aryl, and/or aralkyl groups(carbon number limitation permitting) attached to the aromatic ring oraromatic ring system. Non-limiting examples of heteroarenediyl groupsinclude:

A “heteroarene” refers to the compound H-R, wherein R is heteroaryl.Pyridine and quinoline are non-limiting examples of heteroarenes. Whenthese terms are used with the “substituted” modifier one or morehydrogen atom has been independently replaced by —OH, —F, —Cl, —Br, —I,—NH₂, —NO₂, —CO₂H, —CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃,—NHCH₂CH₃, —N(CH₃)₂, —C(O)NH₂, —OC(O)CH₃, or —S(O)₂NH₂.

The term “acyl” when used without the “substituted” modifier refers tothe group —C(O)R, in which R is a hydrogen, alkyl, cycloalkyl, aryl,aralkyl or heteroaryl, as those terms are defined above. The groups,—CHO, —C(O)CH₃ (acetyl, Ac), —C(O)CH₂CH₃, —C(O)CH₂CH₂CH₃, —C(O)CH(CH₃)₂,—C(O)CH(CH₂)₂, —C(O)C₆H₅, —C(O)C₆H₄CH₃, —C(O)CH₂C₆H₅, —C(O)(imidazolyl)are non-limiting examples of acyl groups. A “thioacyl” is defined in ananalogous manner, except that the oxygen atom of the group —C(O)R hasbeen replaced with a sulfur atom, —C(S)R. The term “aldehyde”corresponds to an alkane, as defined above, wherein at least one of thehydrogen atoms has been replaced with a —CHO group. When any of theseterms are used with the “substituted” modifier one or more hydrogen atom(including a hydrogen atom directly attached the carbonyl orthiocarbonyl group, if any) has been independently replaced by —OH, —F,—Cl, —Br, —I, —NH₂, —NO₂, —CO₂H, —CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃,—C(O)CH₃, —NHCH₃, —NHCH₂CH₃, —N(CH₃)₂, —C(O)NH₂, —OC(O)CH₃, or—S(O)₂NH₂. The groups, —C(O)CH₂CF₃, —CO₂H (carboxyl), —CO₂CH₃(methylcarboxyl), —CO₂CH₂CH₃, —C(O)NH₂ (carbamoyl), and —CON(CH₃)₂, arenon-limiting examples of substituted acyl groups.

The term “alkoxy” when used without the “substituted” modifier refers tothe group —OR, in which R is an alkyl, as that term is defined above.Non-limiting examples of alkoxy groups include: —OCH₃ (methoxy),—OCH₂CH₃ (ethoxy), —OCH₂CH₂CH₃, —OCH(CH₃)₂ (isopropoxy), and —OC(CH₃)₃(tert-butoxy). The terms “cycloalkoxy”, “alkenyloxy”, “cycloalkenyloxy”,“alkynyloxy”, “aryloxy”, “aralkoxy”, “heteroaryloxy”,“heterocycloalkoxy”, and “acyloxy”, when used without the “substituted”modifier, refers to groups, defined as —OR, in which R is cycloalkyl,alkenyl, cycloalkenyl, alkynyl, aryl, aralkyl, heteroaryl,heterocycloalkyl, and acyl, respectively. The term “alkoxydiyl” refersto the divalent group —O-alkanediyl-, —O-alkanediyl-O—, or-alkanediyl-O-alkanediyl-. The terms “alkylthio”, “cycloalkylthio”, and“acylthio” when used without the “substituted” modifier refers to thegroup —SR, in which R is an alkyl, cycloalkyl, and acyl, respectively.The term “alcohol” corresponds to an alkane, as defined above, whereinat least one of the hydrogen atoms has been replaced with a hydroxygroup. The term “ether” corresponds to an alkane, as defined above,wherein at least one of the hydrogen atoms has been replaced with analkoxy or cycloalkoxy group. When any of these terms is used with the“substituted” modifier one or more hydrogen atom has been independentlyreplaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H, —CO₂CH₃, —CN, —SH,—OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃, —NHCH₂CH₃, —N(CH₃)₂, —C(O)NH₂,—OC(O)CH₃, or —S(O)₂NH₂.

The term “alkylamino” when used without the “substituted” modifierrefers to the group —NHR, in which R is an alkyl, as that term isdefined above. Non-limiting examples of alkylamino groups include:—NHCH₃ and —NHCH₂CH₃. The term “dialkylamino” when used without the“substituted” modifier refers to the group —NRR′, in which R and R′ caneach independently be the same or different alkyl groups, or R and R′can be taken together to represent an alkanediyl. Non-limiting examplesof dialkylamino groups include: —N(CH₃)₂, —N(CH₃)(CH₂CH₃), andN-pyrrolidinyl. The terms “alkoxyamino”, “cycloalkylamino”,“alkenylamino”, “cycloalkenylamino”, “alkynylamino”, “arylamino”,“aralkylamino”, “heteroarylamino”, “heterocycloalkylamino” and“alkylsulfonylamino” when used without the “substituted” modifier,refers to groups, defined as —NHR, in which R is alkoxy, cycloalkyl,alkenyl, cycloalkenyl, alkynyl, aryl, aralkyl, heteroaryl,heterocycloalkyl, and alkylsulfonyl, respectively. A non-limitingexample of an arylamino group is —NHC₆H₅. The term “amido” (acylamino),when used without the “substituted” modifier, refers to the group —NHR,in which R is acyl, as that term is defined above. A non-limitingexample of an amido group is —NHC(O)CH₃. The term “alkylimino” when usedwithout the “substituted” modifier refers to the divalent group ═NR, inwhich R is an alkyl, as that term is defined above. The term“alkylaminodiyl” refers to the divalent group —NH-alkanediyl-,—NH-alkanediyl-NH—, or -alkanediyl-NH-alkanediyl-. When any of theseterms is used with the “substituted” modifier one or more hydrogen atomhas been independently replaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂,—CO₂H, —CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃, —NHCH₂CH₃,—N(CH₃)₂, —C(O)NH₂, —OC(O)CH₃, or —S(O)₂NH₂. The groups —NHC(O)OCH₃ and—NHC(O)NHCH₃ are non-limiting examples of substituted amido groups.

B. EXTRACELLULAR PHE

The present disclosure also relates to imaging the extracellular pH(pH_(e)) of a cell or group of cells. In particular, the extracellularenvironment could be of a tumor cell. Aerobic glycolysis (a.k.a. Warburgeffect, FIG. 23A), where cancer cells preferentially take up glucose andconvert it into lactic acids, has rekindled intense interest in imagingpH_(e) of a tumor cell as a method of determine the presence of tumortissue (Heiden et al., 2009). The clinical relevance of the Warburgeffect has already been manifested by the wide clinical use of2-¹⁸F-deoxyglucose (FDG) for tumor diagnosis as well as monitoringtreatment responses. In tumor microenvironment, lactic acids arepreferentially accumulated in the extracellular space due tomonocarboxylate transporters, which are elevated in cancer cellmembranes (Halestrap & Prince 1999). The resulting acidification ofextracellular pH (pH_(e)) in tumors promotes remodeling of extracellularmatrix for increased tumor invasion and metastasis. Recently, Barber andcoworkers described dysregulated pH in tumors as another “hallmark ofcancer” (Webb et al., 2011).

Many previous studies have been performed to quantify the pH_(e) in thetumor microenvironment (Gillies et al., 1994; Gillies et al., 2004; vanSluis et al., 1999 and Volk et al., 1993). FIG. 23B is a representativepH_(e) study in 268 tumors from 30 different human cancer cell lines(Volk et al., 1993). Compared to blood pH (7.4), all the tumor pH_(e)are acidic with an average of 6.84 ranging from 6.71 to 7.01. Althoughthe acidity of tumor pH_(e) is persistent, exploiting it fortumor-specific imaging is challenging due to the relatively small pHdifferences (i.e., <1 pH unit) making probes which possess a very narrowpH transition range of particular interest for this application.

In some embodiments, the present disclosure provides polymers andmicelles which can be used in a pH responsive system that can image andphysiological and/or pathological process that is affected or affectsintracellular or extracellular pH including but not limited toinfections, fistulas, ulcers, ketoacidosis from diabetes or otherdiseases, hypoxia, metabolic acidosis, respiratory acidosis, toxicingestion, poisoning, bone turnover, degenerative diseases, wounds, andtissue damage from burns radiation or other sources.

C. SURGICAL IMAGING OF TUMOR MARGINS

Positive tumor margins, which are defined by the presence of cancercells at the edge of surgical resection, are the most importantindicator of tumor recurrence and survival of HNSCC patients aftersurgery (Woolgar & Triantafyllou 2005; McMahon et al., 2003; Ravasz etal., Atkins et al., 2012 and Iczkowski & Lucia 2011). In someembodiments, any cancer cell line which exhibits a differentextracellular pH environment than the normal physiological pH of theenvironment can be imaged with a pH responsive system disclosed herein.Furthermore, by modifying the dye used in the pH responsive dyes, avariety of different commercially available surgical imaging systems canbe used to measure the margins of the tumor. These systems include butare not limited to systems for open surgery (e.g., SPY Elite®),microsurgery (Carl Zeiss, Leica), laparoscopy (Olympus, Karl Storz), androbotic surgery (da Vinci®). Many of these clinical systems have fastacquisition times allowing real-time imaging during an operation.Furthermore, the mixed polymers disclosed herein as well as ahomopolymer of the any of the individual monomers used to create themixed polymers can be used in the pH responsive system for the imagingof a tumor during an operation.

D. BLOCK COPOLYMERS AND FLUORESCENT DYES

The pH-responsive micelles and nanoparticles disclosed herein compriseblock copolymers and fluorescent dyes. A block copolymer comprises ahydrophilic polymer segment and a hydrophobic polymer segment. Thehydrophobic polymer segment is pH sensitive. For example, thehydrophobic polymer segment may comprise an ionizable amine group torender pH sensitivity. The block copolymers form pH-activatable micellar(pHAM) nanoparticles based on the supramolecular self-assembly of theseionizable block copolymers. At higher pH, the block copolymers assembleinto micelles, whereas at lower pH, ionization of the amine group in thehydrophobic polymer segment results in dissociation of the micelle. Theionizable groups may act as tunable hydrophilic/hydrophobic blocks atdifferent pH values, which may directly affect the dynamic self-assemblyof micelles.

For diagnostic or pH monitoring applications, a labeling moiety may beconjugated to the block copolymer. In some embodiments, the label (e.g.,a fluorescent label) is sequestered inside the micelle when the pHfavors micelle formation. Sequestration in the micelle results in adecrease in label signal (e.g., via fluorescence quenching). Specific pHconditions may lead to rapid protonation and dissociation of micellesinto unimers, thereby exposing the label, and increasing the labelsignal (e.g., increasing fluorescence emission). The micelles of thedisclosure may provide one or more advantages in diagnosticapplications, such as: (1) disassociation of the micelle (and rapidincrease in label signal) within a short amount of time (e.g., withinminutes) under certain pH environments (e.g., acidic environments), asopposed to hours or days for previous micelle compositions; (2)increased imaging payloads; (3) selective targeting of label to thedesired site (e.g., tumor or particular endocytic compartment); (4)prolonged blood circulation times; (5) responsiveness within specificnarrow pH ranges (e.g., for targeting of specific organelles); and (6)high contrast sensitivity and specificity. For example, the micelles maystay silent (or in the OFF state) with minimum background signals undernormal physiological conditions (e.g., blood circulation, cell cultureconditions), but imaging signals can be greatly amplified when themicelles reach their intended molecular targets (e.g., extracellulartumor environment or cellular organelle).

Numerous fluorescent dyes are known in the art. In certain aspects ofthe disclosure, the fluorescent dye is a pH-insensitive fluorescentdyes. In some embodiments, the fluorescent dye is paired with afluorescent quencher to obtain an increased signal change uponactivation. The fluorescent dye may be conjugated to the copolymerdirectly or through a linker moiety. Methods known in the art may beused to conjugate the fluorescent dye to, for example, the hydrophobicpolymer. In some embodiments, the fluorescent dye may be conjugated toamine of the hydrophobic polymer through an amide bond.

Examples of block copolymers and block copolymers conjugated tofluorescent dyes include:

wherein: R₁ is hydrogen, alkyl_((C≤12)), cycloalkyl_((C≤12)),substituted alkyl_((C≤12)), substituted cycloalkyl_((C≤12)), or

or a metal chelating group; n is an integer from 1 to 250; R₂ and R₂′are each independently selected from hydrogen, alkyl_((C≤12)),cycloalkyl_((C≤12)), substituted alkyl_((C≤12)), or substitutedcycloalkyl_((C≤12)); R₃ is a group of the formula:

wherein: X₁, X₂, and X₃ are each independently selected from hydrogen,alkyl_((C≤12)), cycloalkyl_((C≤12)), substituted alkyl_((C≤12)), orsubstituted cycloalkyl_((C≤12)); and X₄ and X₅ are each independentlyselected from alkyl_((C≤12)), cycloalkyl_((C≤12)), aryl_((C≤12)),heteroaryl_((C≤12)) or a substituted version of any of these groups, orX₄ and X₅ are taken together and are alkanediyl_((C≤12)),alkoxydiyl_((C≤12)), alkylaminodiyl_((C≤12)), or a substituted versionof any of these groups; x is an integer from 1 to 100; R₄ is a group ofthe formula:

wherein: X₁′, X₂′, and X₃′ are each independently selected fromhydrogen, alkyl_((C≤12)), cycloalkyl_((C≤12)), substitutedalkyl_((C≤12)), or substituted cycloalkyl_((C≤12)); and X₄′ and X₅′ areeach independently selected from alkyl_((C≤12)), cycloalkyl_((C≤12)),aryl_((C≤12)), heteroaryl_((C≤12)) or a substituted version of any ofthese groups, or X₄′ and X₅′ are taken together and arealkanediyl_((C≤12)), alkoxydiyl_((C≤12)), alkylaminodiyl_((C≤12)), or asubstituted version of any of these groups; y is an integer from 1 to100; R₅ is a group of the formula:

wherein: Y₁, Y₂, and Y₃ are each independently selected from hydrogen,alkyl_((C≤12)), cycloalkyl_((C≤12)), substituted alkyl_((C≤12)), orsubstituted cycloalkyl_((C≤12)); and Y₄ is hydrogen, alkyl_((C≤12)),acyl_((C≤12)), substituted alkyl_((C≤12)), substituted acyl_((C≤12)), adye, or a fluorescence quencher; z is an integer from 0-6; and R₆ ishydrogen, halo, hydroxy, alkyl_((C≤12)), or substituted alkyl_((C≤12)),wherein R₃, R₄, and R₅ can occur in any order within the polymer,provided that R₃ and R₄ are not the same group. In some embodiments,each monomer of R₃, R₄, and R₅ within the longer polymer can occur inany order within the polymer. In some embodiments, the specificcomposition of the polymer (molar fraction of the R₃, R₄, and R₅monomers) is related to the specific pH transition point of thenanoparticle produced using that polymer.

E. MICELLE SYSTEMS AND COMPOSITIONS

The systems and compositions disclosed herein utilize either a singlemicelle or a series of micelles tuned to different pH levels.Furthermore, the micelles have a narrow pH transition range. In someembodiments, the micelles have a pH transition range of less than about1 pH unit. In various embodiments, the micelles have a pH transitionrange of less than about 0.9, less than about 0.8, less than about 0.7,less than about 0.6, less than about 0.5, less than about 0.4, less thanabout 0.3, less than about 0.25, less than about 0.2, or less than about0.1 pH unit. The narrow pH transition range advantageously provides asharper pH response that can result in complete turn-on of thefluorophores with subtle changes of pH.

Accordingly, a single or series of pH-tunable, multicolored fluorescentnanoparticles having pH-induced micellization and quenching offluorophores in the micelle core provide mechanisms for the independentcontrol of pH transition (via polymers), fluorescence emission, or theuse of fluorescence quenchers. The fluorescence wavelengths can be finetuned from, for example, violet to near IR emission range (400-820 nm).Their fluorescence ON/OFF activation can be achieved within no more than0.25 pH units, which is much narrower compared to small molecular pHsensors. In some embodiments, a narrower range for fluorescence ON/OFFactivation can be achieved such that the range is no more than 0.2 pHunits. In some embodiments, the range is no more than 0.15 pH units.Furthermore, the use of a fluorescence quencher may also increase thefluorescence activation such that the difference between the associatedand disassociated nanoparticle is greater than 50 times the associatednanoparticle. In some embodiments, the fluorescence activation isgreater than 75 times higher than the associated nanoparticle Thismulticolored, pH tunable and activatable fluorescent nanoplatformprovides a valuable tool to investigate fundamental cell physiologicalprocesses such as pH regulation in endocytic organelles, receptorcycling, and endocytic trafficking, which are related to cancer,lysosomal storage disease, and neurological disorders.

The size of the micelles will typically be in the nanometer scale (i.e.,between about 1 nm and 1 μm in diameter). In some embodiments, themicelle has a size of about 10 to about 200 nm. In some embodiments, themicelle has a size of about 20 to about 100 nm. In some embodiments, themicelle has a size of about 30 to about 50 nm.

F. TARGETING MOIETIES

The micelles and nanoparticles may further comprise a targeting moiety.The targeting moiety may be used to target the nanoparticle or micelleto, for example, a particular cell surface receptor, cell surfacemarker, or to an organelle (e.g., nucleus, mitochondria, endoplasmicreticulum, chloroplast, apoplast, or peroxisome). Such targetingmoieties will be advantageous in the study of receptor recycling, markerrecycling, intracellular pH regulation, endocytic trafficking.

The targeting moiety may be, for example, an antibody or antibodyfragment (e.g., Fab′ fragment), a protein, a peptide (e.g., a signalpeptide), an aptamer, or a small molecule (e.g., folic acid). Thetargeting moiety may be conjugated to the block copolymer (e.g.,conjugated to the hydrophilic polymer segment) by methods known in theart. The selection of targeting moiety will depend on the particulartarget. For example, antibodies, antibody fragments, small molecules, orbinding partners may be more appropriate for targeting cell surfacereceptors and cell surface markers, whereas peptides, particularlysignal peptides, may be more appropriate for targeting organelles.

G. FLUORESCENCE DETECTION

Various aspects of the present disclosure relate to the direct orindirect detection of micelle disassociation by detecting an increase ina fluorescent signal. Techniques for detecting fluorescent signals fromfluorescent dyes are known to those in the art. For example,fluorescence confocal microscopy as described in the Examples below isone such technique.

Flow cytometry, for example, is another technique that can be used fordetecting fluorescent signals. Flow cytometry involves the separation ofcells or other particles, such as microspheres, in a liquid sample. Thebasic steps of flow cytometry involve the direction of a fluid samplethrough an apparatus such that a liquid stream passes through a sensingregion. The particles should pass one at a time by the sensor and maycategorized based on size, refraction, light scattering, opacity,roughness, shape, fluorescence, etc.

The measurements described herein may include image processing foranalyzing one or more images of cells to determine one or morecharacteristics of the cells such as numerical values representing themagnitude of fluorescence emission at multiple detection wavelengthsand/or at multiple time points.

H. KITS

The present disclosure also provides kits. Any of the componentsdisclosed herein may be combined in a kit. In certain embodiments thekits comprise a pH-responsive system or composition as described above.

The kits will generally include at least one vial, test tube, flask,bottle, syringe or other container, into which a component may beplaced, and preferably, suitably aliquoted. Where there is more than onecomponent in the kit, the kit also will generally contain a second,third or other additional containers into which the additionalcomponents may be separately placed. However, various combinations ofcomponents may be comprised in a container. In some embodiments, all ofthe micelle populations in a series are combined in a single container.In other embodiments, some or all of the micelle population in a seriesare provided in separate containers.

The kits of the present disclosure also will typically include packagingfor containing the various containers in close confinement forcommercial sale. Such packaging may include cardboard or injection orblow molded plastic packaging into which the desired containers areretained. A kit may also include instructions for employing the kitcomponents. Instructions may include variations that can be implemented.

I. SPECT AND PET

Radionuclide imaging modalities (positron emission tomography, (PET);single photon emission computed tomography (SPECT)) are diagnosticcross-sectional imaging techniques that map the location andconcentration of radionuclide-labeled radiotracers. Although CT and MRIprovide considerable anatomic information about the location and theextent of tumors, these imaging modalities cannot adequatelydifferentiate invasive lesions from edema, radiation necrosis, gradingor gliosis. PET and SPECT can be used to localize and characterizetumors by measuring metabolic activity.

PET and SPECT provide information pertaining to information at thecellular level, such as cellular viability. In PET, a patient ingests oris injected with a slightly radioactive substance that emits positrons,which can be monitored as the substance moves through the body. In onecommon application, for instance, patients are given glucose withpositron emitters attached, and their brains are monitored as theyperform various tasks. Since the brain uses glucose as it works, a PETimage shows where brain activity is high.

Closely related to PET is single-photon emission computed tomography, orSPECT. The major difference between the two is that instead of apositron-emitting substance, SPECT uses a radioactive tracer that emitslow-energy photons. SPECT is valuable for diagnosing coronary arterydisease, and already some 2.5 million SPECT heart studies are done inthe United States each year.

PET radiopharmaceuticals for imaging are commonly labeled withpositron-emitters such as ¹¹C, ¹³N, ¹⁵O, ¹⁸F, ⁸²Rb, ⁶²Cu, and ⁶⁸Ga.SPECT radiopharmaceuticals are commonly labeled with positron emitterssuch as ^(99m)Tc, ²⁰¹Tl, and ⁶⁷Ga. Regarding brain imaging, PET andSPECT radiopharmaceuticals are classified according toblood-brain-barrier permeability (BBB), cerebral perfusion andmetabolism receptor-binding, and antigen-antibody binding (Saha et al.,1994). The blood-brain-barrier SPECT agents, such as ^(99m)TcO4-DTPA,²⁰¹Tl, and [⁶⁷Ga]citrate are excluded by normal brain cells, but enterinto tumor cells because of altered BBB. SPECT perfusion agents such as[¹²³I]IMP, [^(99m)Tc]HMPAO, [^(99m)Tc]ECD are lipophilic agents, andtherefore diffuse into the normal brain. Important receptor-bindingSPECT radiopharmaceuticals include [¹²³I]QNE, [¹²³I]IBZM, and[¹²³I]iomazenil. These tracers bind to specific receptors, and are ofimportance in the evaluation of receptor-related diseases.

J. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the disclosure. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the disclosure, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe disclosure.

Example 1: Methods and Materials for Preparation of Library of pHResponsive Nanoprobes

1. Materials

The N-hydroxyl succinimidal (NHS) esters of different fluorophores andfluorescence quenchers were obtained as following: RhoG-NHS, TMR-NHS,ROX-NHS, BDY-NHS, BDY-TMR-NHS, BDY630-NHS, AMCA-x-NHS, MB-NHS, PPO-NHS,QSY35, QSY7 and QSY21 ester were purchased from Invitrogen Company;Cy5-NHS, Cy5.5-NHS, Cy7.5-NHS ester were purchased from LumiprobeCorporation; BHQ-1-NHS ester was purchased from Biosearch Technologies.PEO macroinitiator, MeO-PEO₁₁₄-Br, was prepared from 2-bromo-2-methylpropanoyl bromide and MeO-PEO₁₁₄-OH according to the procedure inBronstein, et al., which is incorporated herein by reference.Bromopropane, bromobutane, bromopentane, ethanolamine, methacrylatechloride and sodium salts were purchased from Sigma-Aldrich. Monomerssuch as 2-(dimethylamino)ethyl methacrylate (DMA-MA),2-(diethylamino)ethyl methacrylate (DEA-MA) and 2-aminoethylmethacrylate (AMA) were purchased from Polyscience Company. AMA wasrecrystallized twice with isopropanol and ethyl acetate (3:7). Monomer2-(dibutylamino) ethyl methacrylate (DBA-MA) was synthesized following apreviously published procedure.² Syntheses of 2-(dipropylamino) ethylmethacrylate (DPA-MA) and 2-(dipentylamino) ethyl methacrylate (D5A-MA)are reported herein. AMA monomer was recrystallized twice withisopropanol and ethyl acetate (3:7) before use. Other solvents andreagents were used as received from Sigma-Aldrich or Fisher ScientificInc. 2. Syntheses of New Methacrylate Monomers

New methacrylate monomers were synthesized following a publishedmethod.[²]Synthesis of 2-(dipropylamino) ethyl methacrylate (DPA-MA) isdescribed here as an example. First, ethanolamine (12.2 g, 0.2 mol) andbromopropane (49.2 g, 0.4 mol) were dissolved in 400 mL acetonitrile,and Na₂CO₃ (53.0 g, 0.5 mol) was added to the solution. After overnightreaction, the solution was filtered to remove the precipitated NaBr saltand extra Na₂CO₃. CH₃CN solvent was removed by rotovap. The resultingresidue was distilled in vacuo (40-45° C. at 0.05 mm Hg) as a colorlessliquid to obtain 2-(dipropylamino) ethanol. Then 2-(dipropylamino)ethanol (21.3 g, 0.1 mol), triethylamine (10.1 g, 0.1 mol), andinhibitor hydroquinone (0.11 g, 0.001 mol) were dissolved in 100 mLCH₂Cl₂ and methacryloyl chloride (10.4 g, 0.1 mol) was added dropwiseinto a three-neck flask. The solution was refluxed overnight. Afterreaction, the solution was filtered to remove the precipitatedtriethylamine-HCl salts, and CH₂Cl₂ solvent was removed by rotovap. Theresulting residue was distilled in vacuo (47-53° C. at 0.05 mm Hg) as acolorless liquid. After synthesis, the monomer was characterized by¹H-NMR. All the NMR spectra were obtained in CDCl₃ usingtetramethylsilane (TMS) as the internal reference on a Varian 500 MHzspectrometer. The characterization of the two new monomers is asfollows:

2-(Dipropylamino) ethyl methacrylate (DPA-MA)

¹H NMR (TMS, CDCl₃, ppm): 6.10 (br, 1H, CHH═C(CH₃)—), 5.54 (br, 1H,CHH═C(CH₃)—), 4.07 (t, 2H, —OCH₂CH₂N—), 3.01 (t, 2H, —OCH₂CH₂N—), 2.68(t, 4H, —N(CH₂CH₂CH₃)₂, 1.94 (s, 3H, CH₂═C(CH₃)—), 1.44 (m, 4H,—N(CH₂CH₂CH₃)₂), 1.01 (t, 6H, —N(CH₂CH₂CH₃)₂)

2-(Dipentylamino) ethyl methacrylate (D5A-MA)

¹H NMR (TMS, CDCl₃, ppm): 6.10 (br, 1H, CHH═C(CH₃)—), 5.55 (br, 1H,CHH═C(CH₃)—), 4.20 (t, 2H, —OCH₂CH₂N—), 2.74 (t, 2H, —OCH₂CH₂N—), 2.45(t, 4H, —N(CH₂CH₂CH₂ CH₂CH₃)₂, 1.94 (s, 3H, CH₂═C(CH₃)—), 1.43 (m, 4H,—N(CH₂CH₂CH₂ CH₂CH₃)₂), 1.30 (m, 4H, —N(CH₂CH₂CH₂ CH₂CH₃)₂), 1.24 (m,4H, —N(CH₂CH₂CH₂ CH₂CH₃)₂), 0.88 (t, 6H, —N(CH₂CH₂CH₂ CH₂CH₃)₂),

2-(Ethylpropylamino) ethyl methacrylate (EPA-MA)

¹H NMR (TMS, CDCl₃, ppm): 6.10 (s, 1H, CHH═C(CH₃)—), 5.54 (s, 1H,CHH═C(CH₃)—), 4.20 (t, 2H, —OCH₂CH₂N—), 2.75 (t, 2H, —OCH₂CH₂N—), 2.58(q, 2H, —N(CH₂CH₂CH₃)(CH₂CH₃)), 2.44 (m, 2H, —N(CH₂CH₂CH₃)(CH₂CH₃)),1.94 (s, 3H, CH₂═C(CH₃)—), 1.45 (m, 2H, —N(CH₂CH₂CH₃)(CH₂CH₃)), 1.02 (t,3H, —N(CH₂CH₂CH₃)(CH₂CH₃)), 0.87 (t, 3H, —N(CH₂CH₂CH₃)(CH₂CH₃))

2-(butyl(isopropyl)amino) ethyl methacrylate (^(ni)D3.5A-MA)

¹H NMR (TMS, CDCl₃, ppm): 6.09 (s, 1H, CHH═C(CH₃)—), 5.53 (s, 1H,CHH═C(CH₃)—), 4.11 (t, 2H, —OCH₂CH₂N—), 2.92 (m, 1H,—N(CH₂CH₂CH₂CH₃)(CH(CH₃)₂), 2.64 (t, 2H, —OCH₂CH₂N—), 2.42 (t, 2H,—N(CH₂CH₂CH₂CH₃)(CH(CH₃)₂), 1.93 (s, 3H, CH₂═C(CH₃)—), 1.38 (m, 2H,—N(CH₂CH₂CH₂CH₃)(CH(CH₃)₂), 1.29 (m, 2H, N(CH₂CH₂CH₂CH₃)(CH(CH₃)₂), 0.97(d, 6H, —N(CH₂CH₂CH₂CH₃)(CH(CH₃)₂), 0.88 (t, 3H,—N(CH₂CH₂CH₂CH₃)(CH(CH₃)₂)

3. Syntheses of PEO-b-PR Block Copolymers

PEO-b-PR copolymers were synthesized by atom transfer radicalpolymerization (ATRP) as described in Zhou, et al., 2011, which isincorporated herein by reference. The dye free copolymers were used inpolymer characterizations. Tables 1-3 summarize the characterization ofeach copolymer. PEO-b-PDPA is used as an example to illustrate theprocedure. First, DPA-MA (1.70 g, 8 mmol), PMDETA (21 μL, 0.1 mmol) andMeO-PEO₁₁₄-Br (0.5 g, 0.1 mmol) were charged into a polymerization tube.Then a mixture of 2-propanol (2 mL) and DMF (2 mL) was added to dissolvethe monomer and initiator. After three cycles of freeze-pump-thaw toremove the oxygen, CuBr (14 mg, 0.1 mmol) was added into thepolymerization tube under nitrogen atmosphere, and the tube was sealedin vacuo. The polymerization was carried out at 40° C. for 8 hours.After polymerization, the reaction mixture was diluted with 10 mL THF,and passed through a neutral Al₂O₃ column to remove the catalyst. TheTHF solvent was removed by rotovap. The residue was dialyzed indistilled water and lyophilized to obtain a white powder.

TABLE 1 Coarse-tuned pH sensitive nanoprobes from Cy5-conjugatedPEO-P(DEA_(x)-D5A_(y)) copolymers. Polymers M_(n) (kDa) M_(w) (Da) PDIYield (%) pH_(t) ΔpH_(10-90%) PD5A 26.9 32.6 1.21 85 4.38 0.19 P(DEA₂₀-21.3 26.3 1.23 90 5.19 0.65 D5A₆₀) P(DEA₄₀- 21.3 25.8 1.20 95 5.99 0.64D5A₄₀) P(DEA₆₀- 22.3 26.4 1.19 90 6.88 0.47 D5A₂₀) PDEA 22.6 26.6 1.1891 7.83 0.14

TABLE 2 Fine-tuned pH sensitive nanoprobes from Cy5-conjugatedPEO-P(DPA_(x)-DBA_(y)) copolymers. M_(n) M_(w) Polymers (kDa) (Da) PDIYield (%) pH_(t) ΔpH_(10-90%) PDBA 22.5 26.8 1.19 80 5.27 0.20P(DPA₂₀-DBA₆₀) 19.7 21.4 1.09 94 5.46 0.19 P(DPA₄₀-DBA₄₀) 21.7 24.7 1.1478 5.70 0.20 P(DPA₆₀-DBA₂₀) 23.9 27.9 1.17 83 5.91 0.18 PDPA 22.6 27.31.21 91 6.21 0.20

TABLE 3 Composition of the UPS library spanning the pH range from 4.4 to7.4. M_(n) Yield Probe Composition (kDa) M_(w) (Da) PDI (%) pH_(t)ΔpH_(10-90%) 4.4 PD5A 26.9 32.6 1.21 85 4.38 0.19 4.7 P(DBA₂₈- 20.2 23.31.15 82 4.67 0.15 D5A₅₂) 5.0 P(DBA₅₆- 20.0 25.9 1.29 84 4.96 0.18 D5A₂₄)5.3 PDBA 22.5 26.8 1.19 80 5.27 0.20 5.6 P(DPA₃₀- 20.4 24.9 1.22 89 5.630.19 DBA₅₀) 5.9 P(DPA₆₀- 23.9 27.9 1.17 83 5.91 0.18 DBA₂₀) 6.2 PDPA20.1 23.3 1.21 91 6.21 0.20 6.5 P(DEA₂₁- 21.8 24.3 1.12 87 6.45 0.19DPA₇₉) 6.8 P(DEA₃₉- 20.3 23.2 1.14 82 6.76 0.20 DPA₆₁) 7.1 P(DEA₅₈- 23.125.2 1.09 85 7.08 0.21 DPA₄₂) 7.4 P(DEA₇₆- 22.5 25.4 1.13 87 7.44 0.18DPA₂₄)

4. Syntheses of PEO-b-(PR-r-Dye/FQ) Block Copolymers

AMA was used for the conjugation of dyes or fluorescence quenchers.Synthesis of PEO-b-(PR-r-AMA) copolymers followed the proceduredescribed above. Three primary amino groups were introduced into eachpolymer chain by controlling the feeding ratio of AMA monomer to theinitiator (ratio=3). After synthesis, PEO-b-(PR-r-AMA) (10 mg) wasdissolved in 2 mL DMF. Then the NHS-ester (1.5 equivalences for Dye-NHSor FQ-NHS) was added. After overnight reaction, the copolymers werepurified by preparative gel permeation chromatography (PLgel Prep 10 m10E3 Å 300×250 columns by Varian, THF as eluent at 5 mL/min) to removethe free dye molecules. The produced PEO-b-(PR-r-Dye/FQ) copolymers werelyophilized and kept at −20° C. for storage.

5. Preparation of Micelle Nanoparticles

Micelles were prepared as has been previously described in Zhou, et al.,2011, which is incorporated herein by reference. In a typical procedure,5 mg of PDPA-Cy5 was dissolved in 0.5 mL THF. Then, the solution wasslowly added into 4 mL of Milli-Q deionized water under sonication. Themixture was filtered 4 times to remove THF using themicro-ultrafiltration system (MWCO=100 KD). Then, the deionized waterwas added to adjust the polymer concentration to 5 mg/mL as a stocksolution. For the mixed micelles, different weight ratios of the PR-Dyeand PR-FQ copolymers were dissolved in 0.5 mL THF, and the sameprocedure was used.

6. Fluorescence Characterization

The fluorescence emission spectra were obtained on a Hitachi fluorometer(F-7500 model). For each copolymer, the sample was initially prepared inMilli-Q water at the concentration of 2 mg/mL. Then the stock solutionwas diluted in 0.2 M citric-phosphate buffers (containing 0.15 M sodiumchloride) with different pH values. The terminal polymer concentrationwas controlled at 100-200 μg/mL.

For the fluorescent images of 4.4-7.1-Cy5s, 5.0-BDY, 5.3-RhoG, 5.6-TMR,5.9-ROX, 6.2-BDY630, 6.5-Cy5, 6.8-Cy5.5 and 7.1-Cy7.5 solutions atdifferent pH values (100 μg/mL for each sample), the Maestro imagingsystem (CRI, Inc., Woburn, Mass.) was used by choosing a proper bandpass excitation filter and a proper long-pass emission filter accordingto the instrument manual. For 4.4-AMCA and 4.7-MB, the images were takenby a camera under the irradiation of a handheld UV light (365 nm). Allmeasurements were conducted at room temperature.

Example 2: Synthesis and Characterization of a Library of pH ResponsivePolymer Micelles

1. Copolymer Syntheses by the ATRP Method.

The atom transfer radical polymerization (ATRP) method (Tsarevsky andMatyjaszewski, 2007; Ma, et al., 2003) with CuBr as a catalyst andN,N,N′,N′,N″-pentamethyldiethylenetriamine (PMDETA) ligand for thecopolymer synthesis (FIG. 1) was used to prepare the copolymers for thestudy. The PEO-b-PR copolymers with homopolymeric PR block weresynthesized using a single metharylate monomer as previously described(Zhou, et al., 2011; Zhou, et al., 2012). In order to continuously finetune the hydrophobicity of the PR segment, a copolymerization strategyusing two methacrylate monomers with different hydrophobicity (FIG. 1)was employed. The molar fraction of the two monomers can be preciselycontrolled prior to polymerization, leading to a random copolymerizedP(R₁-r-R₂) block. A series of methacrylate monomers with differentdialkyl chain lengths (e.g., ethyl, propyl, butyl and pentyl) were usedin the current study. To introduce fluorophores or fluorescencequenchers, aminoethylmethacrylate (AMA-MA) (three repeating units perpolymer chain) was also incorporated where the free amino groups wereconjugated to dyes or FQs through activated N-hydroxyl succinimidyl(NHS) esters.

After syntheses, the copolymers were characterized with ¹H NMR to verifythe chemical compositions, and gel permeation chromatography to measurethe number- and weight-averaged molecular weights and polydispersity(Tables 1-3, FIGS. 2-6).

2. Comparison of Copolymerization Vs. Molecular Mixture Strategy forpH_(t) Control.

Initially, two different strategies on their abilities to control thepH_(t) values of UPS nanoprobes were compared. The first strategyinvolves a molecular mixture of two different PEO-b-PR copolymers withdifferent pH transitions. In this example, Cy5-conjugatedPEO-b-poly[2-(diethylamino)ethyl methacrylate] (PDEA, all the copolymerswere conjugated with Cy5 dye in the PR segment unless specified below)and PEO-b-poly[2-(dipentylamino)ethyl methacrylate] (PD5A) were used.The PDEA and PD5A nanoprobes had pH transitions at 4.4 and 7.8,respectively. A solvent evaporation procedure was used to produce amicelle nanoprobe consisting of both copolymers with the same molarpercentage (i.e., 50%) in each micelle (this was verified by heteroFRETexperiments). In the second strategy, the Cy5-conjugatedPEO-b-poly[2-(diethylamino)ethyl methacrylate-r-2-(dipentylamino)ethylmethacrylate] copolymer (P(DEA₄₀-D5A₄₀)) was synthesized where the PRsegment was composed of a random copolymer from two monomers (40repeating units for each monomer, Table 1). The hydrodynamic diameterswere 65 and 22 nm for PDEA/PD5A (molecular mixture) and P(DEA₄₀-D5A₄₀)(copolymer) micelles, respectively.

The two micelle designs showed a drastically different pattern offluorescence emission vs. pH relationships. For the PDEA/PD5Ananoprobes, distinctive behaviors of pH transitions was observedcorresponding to individual copolymers where the fluorescence on/offtransitions were at 4.4 and 7.8 (FIG. 7A, FIG. 8). This result suggeststhat chain entanglement between PDEA and PD5A within the micelle core isnot sufficient to overcome individual polymer dissociation behaviors. Incontrast, the P(DEA₄₀-D5A₄₀) nanoprobe showed a single pH transition at6.0, about halfway between the PDEA and PD5A transitions.

To explore the control of transition pH, a series of P(DEA_(x)-DSA_(y))copolymers with varying molar fractions of two monomers weresynthesized. The resulting copolymers displayed different pH transitions(FIG. 7B, FIG. 9). Plot of pH_(t) of nanoprobes as a function of themolar fraction of DEA monomer showed a linear correlation (FIG. 7C).Incorporation of higher percentage of less hydrophobic monomers (e.g.,DEA-MA) resulted in higher pH transitions. The transition pH of the UPSnanoprobes can be primarily controlled by varying the hydrophobicity ofthe PR segment. This observation is in contrary to small molecular pHsensors, where electron withdrawing or donating groups are necessary forfine tuning (Urano, et al., 2009).

3. Monomer Compatibility Affects Sharpness of pH Transition.

Although P(DEA_(x)-D5A_(y)) nanoprobes with different monomer percentageallowed control of transition pH (FIGS. 7B-C), the sharpness of pHtransitions was significantly broader than the corresponding nanoprobeswith homopolymeric PR segment. More specifically, the ΔpH₁₀₋₉₀% values(the pH range where fluorescence intensity increases from 10 to 90%)were 0.65, 0.64, and 0.47 for P(DEA_(x)-D5A_(y)) copolymers with 25, 50and 75% of DEA-MA compositions, respectively, in comparison to 0.14 and0.19 for PDEA and PD5A nanoprobes, respectively. The broad pH responsefrom P(DEA_(x)-D5A_(y)) copolymers indicates the heterogeneous chainproperty from the monomers with large hydrophobicity differences.

To improve the sharpness of pH transition, the use of monomers withclosely matched hydrophobicity were investigated. As an example,2-(dipropylamino)ethyl methacrylate (DPA-MA) and 2-(dibutylamino)ethylmethacrylate (DBA-MA) was chosen to produce a series ofP(DPA_(x)-DBA_(y)) nanoprobes. The two monomers differ by one carbon onthe nitrogen substituents (i.e., propyl vs. butyl). Copolymerization ofthe two monomers led to a more refined, tunable series of nanoprobeswith sharp pH transitions (FIG. 10A, FIG. 11). The ΔpH₁₀₋₉₀% values were0.19, 0.20, and 0.18 for P(DPA_(x)-DBA_(y)) nanoprobes with 25, 50 and75% of DPA-MA compositions, respectively. Each copolymer probemaintained the sharp pH transition (<0.25 pH unit). FIG. 10B shows afluorescence derivative plot as a function of pH, which furtherillustrates the greatly increased sharpness of serial P(DPA_(x)-DBA_(y))nanoprobes compared to a single P(DEA₄₀-D5A₄₀) nanoprobe in the same pHspan.

Plot of pH_(t) values of the P(DPA_(x)-DBA_(y)) nanoprobes as a functionof molar fraction of DPA-MA monomer also yielded a linear correlation(FIG. 10C). Similarly, standard curves for P(DBA_(x)-D5A_(y)) andP(DEA_(x)-DPA_(y)) series were established demonstrating linearrelationships between pH_(t) and molar fraction of the monomers. Thesestandard curves allow for the rational design of UPS nanoprobes with anypredetermined pH transitions (between 4.4-7.8) by choosing copolymerswith correct PR compositions (i.e., selection of monomer pairs andspecific molar fractions). For proof of concept, a UPS libraryconsisting of 10 nanoprobes with 0.3 pH increment covering the entirephysiologic range of pH (4.4-7.4) were generated while each nanoprobemaintained the sharp pH transitions (<0.25 pH unit between on and offstates, FIG. 10D, FIGS. 12-13).

4. Use of Fluorescence Quenchers to Broaden Fluorophore Selection.

Previously, homo-FRET induced fluorescence decay is the main mechanismto achieve the on/off activatable design of the UPS nanoprobes wasreported (Zhou, et al., 2012). This mechanism only applies tofluorophores (e.g., rhodamine and cyanine dyes) with small Stoke shifts(<40 nm). For dyes with large Stoke shifts (e.g., marina blue or PPO,Δλ≥100 nm), the fluorescence activation ratio (R_(F)=F_(on)/F_(off),where F_(on) and F_(off) are the fluorescence intensity at on and offstates, respectively) was less than 5. Moreover, for BODIPY® families ofdyes, the pH transition was broad (>0.5 pH unit) with relatively lowR_(F) (<15) as a result of the photo-induced electron transfer (PeT)mechanism (Petsalakis, et al., 2008; Tal, et al., 2006; Dale and Rebek,2006)

To overcome these limitations, the use of fluorescence quenchers (FQs)to broaden the fluorophore selection was investigated. Fluorescencequenchers have been widely used by many groups for the design ofactivatable imaging probes (Blum, et al., 2005; Lee, et al., 2009; Levi,et al., 2010; Maxwell, et al., 2009). The mechanism is based on thefluorescence resonance energy transfer from desired fluorophores to theFQs, which subsequently dissipate the radiative energy into heat. Inthis design, a series of FQs that are sensitive to different emissionwavelengths were prepared and conjugated onto the copolymer (FIG. 14).The UPS nanoprobes were produced by mixing the FQ-conjugated polymerwith dye-conjugated polymer in the same micelle core. At the micellestate, the FQs are anticipated that the compounds would effectivelyquench the fluorophore signals and upon micelle dissociation, separationof FQs and fluorophores will result in significant increase influorescence emissions (FIG. 15A).

To evaluate the effectiveness of the FQ strategy,PEO-b-poly[2-(propylamino)ethyl methacrylate] (PDPA) were used as amodel system and different FQs and fluorophores were conjugated to thecopolymer. The PDPA nanoprobe had a pH transition at 6.2. First, the FQstrategy on fluorophores with large Stoke shift (e.g., AMCA: 353/442;marina blue or MB: 362/462; PyMPO or PPO: 415/570 was investigated. Thetwo numbers refer to the excitation and emission wavelengths,respectively). Without the introduction of FQ-conjugated polymer, thePDPA-AMCA and PDPA-MB nanoprobes showed only 3-fold fluorescenceactivation between the on and off states at pH 5.0 and 7.4, respectively(FIG. 18A). Introduction of PDPA-QSY35 to PDPA-AMCA or PDPA-MB resultedin significant increase in fluorescence activation, which reached aplateau when the molar fraction of PDPA-QSY35 became 67% (FIG. 16A). Atthis composition, the R_(F) values reached approximately 90-fold, whichare 30 times higher than those without the FQs (FIG. 18B). Similarly,introduction of PDPA-QSY7 (50 mol %) to PDPA-PPO nanoprobes increasedthe R_(F) value from 6 to >130-fold, respectively (FIG. 18B).

For BODIPY® families of dyes, the PDPA-BDY493 and PDPA-TMR nanoprobesonly yielded ˜15-fold of fluorescence activations (FIG. 18C), which arenot adequate in biological applications (e.g., during cellular imaging,an R_(F) value >30 is necessary to suppress the background signals).Introduction of PDPA-BHQ1 (50 mol %) and PDPA-QSY7 (50 mol %) to thePDPA-BDY493 and PDPA-TMR nanoprobes led to dramatically increased R_(F)values (both >100-fold, FIG. 18D, FIG. 19). Interestingly, PDPA-BDY630alone was able to achieve a 40-fold R_(F) value. Addition of PDPA-QSY21further increased the R_(F) value to over 250-fold (FIG. 18D)

Previous studies showed that rhodamine and cyanine dyes with small Stokeshifts (<40 nm) were able to produce UPS nanoprobes with large R_(F)values through the homoFRET-induced fluorescence decay mechanism (Zhou,et al., 2012). Results from this study confirmed the previous report,where PDPA-dye copolymers alone reached >50-fold and >100-fold forrhodamine and cyanine dyes, respectively. Addition of FQ-conjugatedcopolymer further increased the RF values for these nanoprobes (FIG.18F, FIGS. 20-22).

FIGS. 18E-F summarized the fluorescence activation ratios(R_(F)=F_(5.0)/F_(7.4)) for all the fluorophores used in PDPA nanoprobeswith and without the introduction of fluorescence quenchers. Data showthat with the addition of FQ-conjugated polymer, all the fluorophores(12 in total) showed universally high activation ratios (>50-fold)regardless of the Stoke shift or PeT mechanisms. In addition,introduction of FQ-conjugated polymer did not affect the sharpness of pHtransitions (all the composite nanoprobes had <0.25 pH unit between onand off states, FIGS. 18B and 18D and FIGS. 22B-22D).

5. UPS Library Spanning Large pH Transitions and Fluorescence Emissions.

Based on the above results, a representative UPS library consisting of10 nanoprobes each encoded with a different fluorophore was produced.The composition for each nanoprobe follows that from FIG. 10D (see Table3 for details), which resulted in a collection with 0.3 pH increment inthe pH span of 4 to 7.4. For each nanoprobe, a series of aqueoussolutions of the copolymer at the same polymer concentration (i.e., 0.1mg/mL) but different pH values were prepared. For 4.4-AMCA, 4.7-MB,5.0-BDY and 6.2-BDY630 nanoprobes, the corresponding copolymers weremixed with the same equivalent of FQ-conjugated matching copolymers toachieve high on/off contrast. FIG. 24 shows the emission image of theUPS nanoprobe library at the excitation/emission wavelengthscorresponding to each fluorophore.

Results from FIG. 24 illustrate the exquisite pH sensitivity of the UPSnanoprobes to the external environment spanning the entire physiologicpH of 4-7.4. In the lowest pH range, the 4.4-AMCA nanoprobe was off atpH 4.55 but can be turned on at pH 4.25. This nanoprobe can be useful inthe detection of functional lysosomal pH where hydrolases require alower pH for enzyme activity. The on/off characteristics of thenanoprobe make them particularly useful in high through screeningapplications to identify molecular pathways or small molecularperturbators that affect lysosomal functions. For the nanoprobescovering the higher pH range (e.g., 6.5-7.1), the nanoprobes can beuseful for the imaging of the acidic pH_(e) of tumors and correlatenanoprobe activation with glycolysis rates of the cancer cells (Wang, etal., 2014; Ko, et al. 2010). The nanoprobes in the intermediate range(e.g., 5.0-6.5) may be useful for the study of maturation ofendosomes/lysosomes and establish organelle-specific compositions forsubcellular imaging or drug delivery applications.

Example 3: Anion Driven Micelle Formation Methods

1. Syntheses of PEO-b-PR Block Copolymers

PEO-b-PR copolymers (Scheme 1) were synthesized by atom transfer radicalpolymerization (ATRP) as reported by Zhou, et al., 2011, which isincorporated herein by reference. The dye-free copolymers were used inpolymer characterizations. PEO-b-PDPA (3) is used as an example toillustrate the procedure. First, DPA-MA (1.70 g, 8 mmol), PMDETA (21 μL,0.1 mmol) and MeO-PEO₁₁₄-Br (0.5 g, 0.1 mmol) were charged into apolymerization tube. Then a mixture of 2-propanol (2 mL) and DMF (2 mL)was added to dissolve the monomer and initiator. After three cycles offreeze-pump-thaw to remove oxygen, CuBr (14 mg, 0.1 mmol) was added intothe polymerization tube under nitrogen atmosphere, and the tube wassealed in vacuo. The polymerization was carried out at 40° C. for 8hours. After polymerization, the reaction mixture was diluted with 10 mLTHF, and passed through a neutral Al₂O₃ column to remove the Cucatalyst. The THF solvent was removed by rotovap. The residue wasdialyzed in distilled water and lyophilized to obtain a white powder.Table 4 summarizes the characterization of each copolymer.

TABLE 4 Characterization of PEO-b-(PR-r-AMA) block copolymers. Repeatingunits Yield M_(w,GPC) M_(n,GPC) In the PR M_(n,1H NMR) Copolymer PR name(%) (×10⁻⁴ D)^(a) (×10⁻⁴ D)^(a) PDI^(a) block (×10⁻⁴ D)^(b) 1 PDMA 862.28 1.87 1.22 92 1.99 2 PDEA 87 2.42 1.97 1.23 88 2.17 3 PDPA 88 2.452.06 1.19 80 2.25 4 PDBA 78 2.84 2.32 1.22 72 2.47 5 PD5A 72 3.11 2.581.20 82 2.75 ^(a)Number-average (Mn), weight-average molecular weight(Mw) and polydispersity index (PDI) (PDI = Mw/Mn) were determined by GPCusing THF as the eluent. ^(b)Determined by ¹H-NMR.

2. Syntheses of PEO-b-(PR-r-TMR/Cy5) Block Copolymers

AMA monomer was incorporated in the copolymers for the conjugation ofdyes (Scheme S1b). Synthesis of PEO-b-(PR-r-AMA) copolymers followed theprocedure described above. Three primary amino groups were introducedinto each polymer chain by controlling the feeding ratio of AMA monomerto the initiator (ratio=3). In a representative procedure,PEO-b-(PR-r-AMA) (50 mg) was dissolved in 2 mL DMF. Then the NHS-ester(2.0 equivalence for TMR-NHS and 1.0 equivalence for Cy5-NHS) was added.After overnight reaction, the copolymers were purified by preparativegel permeation chromatography (PLgel Prep 10 m 10E3 Å 300×250 columns byVarian, THF as eluent at 5 mL/min) to remove the free dye molecules. Theproduced PEO-b-(PR-r-Dye) copolymers were lyophilized and kept at −20°C. during storage. It is important to note that the dye would undergoboth Hetero FRET as well self-quenching when block copolymersself-assembled into micelles. So the dye conjugation number for eachpolymer chain is important for the FRET experiment. In the experiment,the conjugation number of TMR and Cy5 was controlled at 2 and 1 perpolymer chain, respectively.

3. Preparation of Ricelle Nanoparticles

For each copolymer, the stock solution of micelles was preparedfollowing a solvent evaporation method as described in Nasongkla, et al.(2006), which is incorporated herein by reference. In the example ofPEO-b-(PDPA-r-TMR) micelle solution, 20 mg of the copolymer was firstdissolved in 1.0 mL THF and then added into 8 mL deionized waterdropwise under sonication. The THF was removed through ultrafiltrationwith (100 KD) membrane for five times. Then deionized water was added toadjust the polymer concentration to 5 mg/mL as a stock solution.PEO-b-PDMA stock solution could be made by directly dissolve copolymerin deionized water.

Micelle solution samples for FRET experiment were prepared in a similarmethod. Preparation of PEO-b-(PDPA-TMR/Cy5) samples was described as arepresentative procedure. First, 0.1 mL PDPA-TMR and 0.1 mL PDPA-Cy5stock solution was added to 1.8 mL deionized water. Then 1.8 μL of 1.0 MHCl was added to dissolve the water-insoluble block copolymer and adjustsolution pH to 4. The Cl⁻ from HCl in the starting sample was <2 mM,which could be neglected for their ability to perturb micellizationaccording to the experimental results.

4. FRET Experiment

The fluorescence emission spectra were obtained on a Hitachi fluorometer(F-7500 model). The samples were excited at 545 nm, and the emissionspectra were collected from 560 to 750 nm. The FRET experiment forPEO-b-PDPA self-assembly behavior with the introduction of differentanions followed similar procedure. ClO₄ ⁻ was used as an example: 0.2 μLof 10 M NaClO₄ solution was added to 2.0 mL 0.5 mg/mL dye-conjugatedPDPA (PDPA-TMR/PDPA-Cy5=1:1) solution at pH=4 and adjusted the ClO₄ ⁻concentration to 1 mM. Then small volume of 10 M NaClO₄ solution wasadded incrementally to increase the ClO₄ ⁻ concentration to 3.2, 5.6, 10mM. After 10 mM, solid NaClO₄ was added to the solution to increase theClO₄ ⁻ concentration to avoid sample dilution. The total volume of addedNaClO₄ is less than 2 μL, which can be neglected compared to totalvolume of 2 mL. The fluorescence emission spectrum was collected after 4min vortex following each addition of NaClO₄.

5. TEM and DLS Characterization

Samples for TEM and DLS analyses were prepared following proceduresdescribed above. The transition pH of PEO-b-PDPA was 6.1. First, 0.1 mLPDPA-TMR and 0.1 mL PDPA-Cy5 stock solution was added to 1.6 mLdeionized water. Solid NaClO₄ and NaCl were then added to the solutionand dissolved after vortex. HCl and NaOH solution (1 M) were used toadjust the solution pH to 5.0 and 7.4. Deionized water was added toadjust the total volume to 2 mL. The morphology and size ofnanoparticles were characterized by transmission electron microscopy(TEM, JEOL 1200EX model). Hydrodynamic diameter (D_(h)) was determinedby dynamic light scattering (DLS, Malvern MicroV Model, He-Ne Laser,λ=632 nm).

6. Anion Competition Experiment

The preparation of micelle samples followed the same proceduresdescribed in FRET experiment. Solid NaCl and Na₂SO₄ powders weredissolved in the aqueous solution to achieve the initial anionconcentration. The initial concentrations of Cl⁻ were 0, 50, 100, 200,500, 1000 and 2000 mM. The initial concentrations of SO₄ ²⁻ were 0, 25,50, 100, 200 and 500 mM. The fluorescence emission spectra werecollected 4 mins after vortex following the addition of NaClO₄. Theresults were fit with a sigmoidal curve. The half maximal FRETefficiency concentration of perchlorate was defined as FC₅₀ to quantifythe competition ability of Cl⁻ and SO₄ ²⁻.

7. ClO₄ ⁻ Induced Micelle Self-Assembly of PEO-b-PR Copolymers

A series of PEO-b-PR_(copolymers) (1-5 in FIG. 25) with different alkylside chains were used in this study. The preparation of micelle samplesfollowed that described in the FRET experiment section. In this seriesof experiments, the ionic strength of the solution was buffered by usinga 100 mM of NaCl concentration. This was used to minimize the ionicstrength contribution from NaClO₄ since more hydrophobic PEO-b-PRcopolymer (e.g., 5) requires less concentration to induce micelleself-assembly. After the experiments, the FRET efficiency was calculatedas previously described in the FRET section.

Example 4: Anion Driven Micelle Formation Results and Discussion

The discovery of the surprising chaotropic anion-induced micellizationof protonated PEO-b-PR copolymers at pH below pH_(t) (FIG. 25) isdescribed. Surprisingly, an anti-Hofmeister trend was observed, wherechaotropic anions resulted in micellization but not the kosmotropicanions (Zhang and Cremer, 2006; Parsons, et al., 2011; Kunz, et al.,2004), in contrary to their effects in protein aggregation (FIG. 26A).

First, a fluorescence energy resonance transfer (FRET) method toinvestigate the micelle self-assembly process was established. FRET ishighly sensitive in detecting conformational and phase transitions ofpolymers/proteins because the energy transfer efficiency is inverselyproportional to the sixth power of the donor-acceptor distance(Jares-Erijman and Jovin, 2003; Sapsford, et al., 2006). In the method,block copolymers were conjugated (1-5 in FIG. 25, Table 4) (Tsarevskyand Matyjaszewski, 2007; Ma, et al., 2003) with either a donor oracceptor dye. PEO-b-poly(dipropylaminoethyl methacrylate) (3,pH_(t)=6.1) was chosen as a model copolymer, and tetramethyl rhodamine(TMR, λ_(ex)/λ_(em)=545/580 nm)/Cy5 (λ_(ex)/λ_(em)=647/666 nm) asdonor/acceptor, respectively (Ha, et al., 1999; Grunwell, et al., 2001)

At pH 4, the tertiary amines in 3 (pH_(t)=6.1) were protonated and theresulting copolymers were soluble in water as dispersed cationicunimers. No FRET effect was observed due to the large distance betweenthe unimers (therefore TMR and Cy5) in solution. Addition of chaotropicanions (e.g., ClO₄ ⁻, SCN⁻ or I⁻) resulted in the decrease offluorescence intensity from TMR and increase of emission intensity ofCy5 (FIG. 27), indicating the formation of polymeric micelles. Micelleformation was hypothesized to bring TMR and Cy5 to close proximitywithin the micelle core, thereby dramatically increasing FRET efficiency(FIG. 26B). In contrary, kosmotropic anions (e.g., SO₄ ²⁻, H₂PO₄ ⁻) didnot lead to any FRET transfer (FIG. 28) even at concentrations close totheir solubility limits (Table 5).

TABLE 5 Saturated solubility of sodium salts of Hofmeister anions. SaltSaturated solubility (M) Salt Saturated solubility (M) NaClO₄ 17.2 NaBr8.8 NaSCN 17.1 NaCl 6.1 NaI 11.9 NaH₂PO₄ 7.2 NaNO₃ 5.0 Na₂SO₄ 1.4Solubility data were obtained from solubility handbook by Khaled Gharibfrom open sources: [1] srdata.nist.gov/solubility/index.aspx [2]food.oregonstate.edu/learn/sugar.html [3] world-wide-web atkayelaby.npl.co.uk/ [4] chemfinder.cambridgesoft.com

The FRET effects were quantified to compare different anions in theirabilities to induce micellization (FIG. 26C). FRET efficiency wasnormalized as (FA/FD)/(FA/FD)_(max), where FA and FD were thefluorescence intensity of TMR and Cy5 at different anion concentrations,respectively; (FA/FD)_(max) was the maximum value of FA/FD (3.3) at highClO₄ ⁻ concentrations. FRET efficiency was plotted as a function ofconcentration for different anions. Results displayed an anti-Hofmeistertrend where chaotropic anions were able to induce unimer association(i.e., micellization) whereas the kosmotropic anions were not (FIG.26C). This observation is in contrary to the classical Hofmeister effectin protein solubilisation, where kosmotropic ions are known to induceprotein aggregation in water but not the chaotropic ions (Hofmeister,1888; Collins and Washabaugh, 1985)

Copolymer 3 displayed different detection sensitivity toward thechaotropic anions. Data show FRET sensitivity followed the order of ClO₄⁻>SCN⁻>I⁻>NO₃ ⁻. FC₅₀ is defined as the anion concentration that theFRET efficiency was at 50%. The values of FC₅₀ were 11, 68 and 304 mMfor ClO₄ ⁻, SCN⁻, and I⁻, respectively. For NO₃-, only weak FRET effectwas observed at its saturation concentration (˜3 M). More detailedexamination shows that only 3-fold ClO₄ ⁻ concentration change (i.e.,from 6 to 18 mM, FIG. 26C) was necessary to increase FRET efficiencyfrom 10% to 90%. This narrowed concentration dependence suggests anincreased cooperative response similar to the ultra-pH response asreported previously (Zhou, et al., 2011; Zhou, et al., 2012; Huang, etal., 2013; Wang, et al., 2013).

To further confirm chaotropic anion-induced micellization, transmissionelectron microscopy (TEM) and dynamic light scattering (DLS) wasemployed to investigate the changes in morphology and hydrodynamicdiameter during micelle transition, respectively. The chloride anion(Cl⁻) was used as a negative control. In the presence of 50 mM Cl⁻,copolymer 3 stayed as a unimer at pH 5.0 (below its pH_(t) at 6.1, FIG.29A). In contrast, copolymer 3 self-assembled into spherical micelleswhen Cl⁻ was replaced with ClO₄ ⁻ (FIG. 29B). DLS analyses showedincrease of hydrodynamic diameters from 7±2 to 26±3 nm when the anionswere changed from Cl⁻ to ClO₄, respectively (FIG. 29). This sizeincrease reflects the transition of copolymer 3 from unimer state to themicelle state, consistent with the FRET and TEM data. At pH 7.4,copolymer 3 was present as spherical micelles with hydrodynamicdiameters at 27±2 and 28±3 nm in the presence of Cl⁻ and ClO₄ ⁻ anions,respectively (FIGS. 30-31). For non-ionizable amphiphilic blockcopolymers such as PEO-b-poly(D,L-lactic acid) (PEO-b-PLA), neither pHchange nor ClO₄ ⁻ addition had any effects on the micelle state (FIG.32).

The chaotropic anion-induced self-assembly were then studied in thepresence of competing kosmotropic or borderline anions. Copolymer 3 wasdissolved at pH 4 with different initial concentrations of competing SO₄²⁻ or Cl⁻. Then chaotropic anions ClO₄ ⁻ were added to inducemicellization (FIGS. 33-36). FIG. 37A shows the representative exampleof FRET efficiency as a function of ClO₄ ⁻ concentration. Addition ofSO₄ ²⁻ anions was able to decrease the sensitivity of ClO₄ ⁻ in micelleinduction. The FC₅₀ values were quantified to evaluate the effect ofcompeting anions (FIG. 37B). A bell curve as a function of the ionicstrength of the competing anions was observed. At low ionic strength(<0.1 M), addition of competing anions decreased the ability of ClO₄ ⁻to induce micelle formation, consistent with their competition with theammonium groups of the PR segment. At high ionic strength (>0.5 M) ofSO₄ ²⁻ or Cl⁻, however, an enhancement of ClO₄ ⁻ induced self-assemblywas observed. This effect can be attributed to the more ordered bulkwater structures at high kosmotropic ion concentrations, which makes thehydrophobic association during micelle self-assembly more favorable.

Finally, the effect of hydrophobic strength of PR segment on chaotropicanion-induced micelleization (FIG. 38A) was investigated. A series ofPEO-b-PR copolymers (1-5 in FIG. 25) bearing different alkyl chainlengths from methyl to pentyl groups on the tertiary amines weresynthesized. Results showed a clear dependence of ClO₄ ⁻-inducedself-assembly on the hydrophobicity of the PR segment (FIG. 39). Withthe least hydrophobic side chains (i.e., methyl in 1), no micellizationwas observed even at the highest ClO₄ ⁻ concentrations (1 M). Incontrary, the most hydrophobic side chains (pentyl in 5) resulted in themost sensitive micellization induction by ClO₄ ⁻. The FC₅₀ values were2, 4, 35, 134 mM when the side chains were pentyl, butyl, propyl andethyl groups, respectively (FIG. 38A).

Results from the above studies illustrate a highly unusual micelleself-assembly process from block copolymers with tertiary ammoniumgroups induced by chaotropic anions. There are several unique featuresin the current nanosystem: first, chaotropic anions were able to formstable ion pairs with positively charged ammonium groups in thehydrophobic micelle core environment. Assuming majority of the ammoniumgroups are in the ionized state, this translates into ˜60,000 ion pairsper micelle with an estimated core size of 14 nm (calculation based on800 polymer chains per micelle, (Wang, et al., 2013) 70-80 repeatingunits of amino group-containing monomers per polymer chain and PEO shellsize of 6 nm) (Leontidis, 2002). Second, only chaotropic anions wereable to induce micelle formation whereas the kosmotropic (SO₄ ²⁻) andborderline (Cl⁻) anions did not posses this ability. This trend appearsto counter that in classical protein solublization studies. Third, theability of chaotropic anions to induce micellization appears to showpositive cooperativity similar to ultra-pH sensitive response. Previousstudies had showed that fluorescence activation (10% to 90% response)occurred within 0.25 pH unit (<2-fold in [H⁺]). This study show FRETtransfer happened in a span of 3-fold [ClO₄ ⁻ ] change. Lastly,competition experiments with kosmotropic and borderline anionsillustrated a bell curve behavior, which points to the complexity andsubtle nature of the micelle self-assembly process in the currentsystem.

An empirical model (FIG. 38B) was built to depict the factors thatcontribute to the micelle self-assembly process. Without being bound bytheory, the hydrophobic interactions from increasing alkyl chain lengthsare hypothesized to provide the dominant driving force for micelleformation. This is supported by the lack of micelle formation when theside chain of the tertiary amines is methyl group (as indicated by thedashed line on the left arm of FIG. 38B). Similarly, neutralizedcopolymer 1 did not form micelles at pH above its pH_(t) (Zhou, et al.,2011) Meanwhile, anions also play a critical role in micellization.Kosmotropic anions, which are known to have strong hydration shells andweak polarization characteristics are energetically less favorable inthe formation of ion pairs and stabilization of ion pairs in thehydrophobic core (Collins, 1997; Underwood and Anacker, 1987).Chaotropic anions, with their strong polarizability and low energy costat removing hydration sheath allows for formation of stable ion pairs inthe hydrophobic micelle core (Zhang and Cremer, 2009).

Example 5: Sentinel Lymph Node Detection

1. Identification of at Risk Sentinel Lymph Nodes by UPS6.9

The UPS_(6.9) nanoprobes also demonstrated the ability to identify atrisk sentinel lymph nodes. FIG. 42A shows the identification of arepresentative sentinel lymph node on the side of the neck near theprimary tumor site by the SPY Elite® camera. Eight lymph nodes wereidentified in 4 different animals (2 per animal) with primary head andneck cancers. These nodes were in the cervical basin draining theprimary head and neck tumors in the mice and anatomically correspondedto cervical nodes typically found in mice. All the eight nodalstructures were identified by UPS_(6.9) only; they were too small to beseen with white light being a millimeter or less in size and closelyassociated with cervical fat and salivary glands but were bright whenvisualized with the SPY camera. H&E analysis by a clinical pathologistvalidated the identified structures as lymph nodes. One out of eightnodes showed the presence of HN5 cancer cells, as indicated by the blackarrows in FIG. 42B bottom panel. In several cases, nodal recurrence oftumors was observed in mice that had had complete resection of theirprimary tumors. Large tumors appeared in the side of the neck instead ofthe primary tumor site. These data suggest the importance ofidentification of at risk lymph nodes to achieve complete resection ofthe tumors. The fact that single nodes draining the tumors wereidentified, and that the majority did not contain cancer cells suggests,these nodes represent SLN collecting activated polymer probes draininginto lymphatics from the primary tumor sites.

Example 6: Development of pH-Activated Indocyanine Green-EncodedNanosensor (PINS)

1. Preparation of PINS and Nanosensor Characteristics

A pH-activatable indocyanine green-encoded nanosensor (PINS) comprisinga micelle of poly(ethylene glycol)-b-poly(ethylpropylaminoethylmethacrylate) copolymers (PEG-b-(PEPA_(x)-r-ICG_(y)), where x and yindicate the number of random repeating units of EPA monomer and ICGdye, respectively; FIGS. 43A-43I) was synthesized. Hydrophobicmicellization and homoFRET-induced fluorescence quenching (Zhou et al.,2011 and Zhou et al., 2012) rendered dramatically sharpened pH response.Systematic optimization of PEPA segment length and ICG conjugationnumber (FIGS. 44A-44F) led to an optimal PINS composition with sharp pHtransition at 6.9, high fluorescence activation ratio, optimal particlesize (25 nm), and an average of 800 ICG per nanoprobe for signalamplification. Compared to reported pH-sensitive probes (e.g., smallmolecular dyes, (Urano et al., 2009) peptides, (Weerakkody et al., 2013)or PeT nanoprobes (Diaz-Fernandez et al., 2006) with 10-fold signalchange over 2 pH), the PINS design achieved >100-fold signal increaseover 0.15 pH span at 6.9. Additional polymers linked to ICG dye wereprepared and are characterized in Table 6.

TABLE 6 Mixed Alkyl Monomer Co-polymers Characteristics. M_(n) M_(w)Polymers (kDa) (Da) PDI Yield (%) pH_(t) ^(a) ΔpH_(10-90%) P(DEA₂₂- 20.726.4 1.28 86 7.10 0.15 EPA₇₈) P(DEA₁₁- 21.1 27.1 1.28 88 7.01 0.16EPA₈₉) PEPA₁₀₀ 21.2 26.6 1.25 87 6.92 0.15 P(DPA₁₀-EPA₉₀) 20.5 24.8 1.2183 6.82 0.14 P(DPA₂₁-EPA₇₉) 20.9 25.9 1.24 82 6.72 0.16 ^(a)pH_(t) wasdetermined by the titration curve of Probe-ICG

Initial dose-response study with the nanosensor was performed in micebearing human head and neck HN5 orthotopic tumor xenografts similar tothose due with other pH responsive systems described herein. The PINSwas intravenously injected through the tail vein and a clinical SPYElite® camera was used to image the animals (FIGS. 45A-45E). The 2.5mg/kg amount was chosen for use as the imaging dose due to the largetumor contrast over noise ratio (CNR=27) over a persisted time window(12-24 h). The stable time window is advantageous for oncologic surgeryover small molecular tracers with transient windows (2-3 h) (Choi etal., 2013) due to fast renal clearance. Injection of free ICG at theequivalent dye dose as in 2.5 mg/kg PINS showed no observable tumorcontrast (FIG. 45B).

Tumor acidosis imaging by PINS improved sensitivity and specificity oftumor detection compared to FDG-PET where brain and brown adiposetissues led to false positives mimicking clinical observations (FIG. 46Band FIGS. 47A-47E) (Cook et al., 2004 and Fukui et al., 2005). AlthoughFDG-PET detected large HN5 tumors (˜200 mm³), the PET method was notsuccessful at detecting small tumor nodules (˜15 mm³, Table 7). Multipledifferent tumor sizes were detectable using PINS with high tumor tonormal tissue contrast (CNR>20). Furthermore, PINS was able to delineatetumor margins at submillimeter spatial resolutions (FIG. 46B and FIGS.47A-47E).

TABLE 7 Characterization of PEPA_(x)-ICG₁ copolymers with differentrepeating units of PEPA segment but the same ICG content and theresulting nanoprobe properties. Re- M_(n) peat Particle copolymer(kDa)^(a) unit^(b) size (nm) pH_(t) ΔpH_(10-90%) FI_(HS6.0) ^(c)PEPA₄₀-ICG₁ 13.5 43 21.9 ± 1.7 6.96 0.30 32.3 PEPA₆₀-ICG₁ 16.8 62 24.8 ±0.9 6.94 0.25 37.0 PEPA₈₀-ICG₁ 19.7 79 25.3 ± 0.8 6.92 0.18 45.3PEPA₁₀₀-ICG₁ 25.1 102 26.0 ± 1.1 6.92 0.15 49.3 PEPA₁₂₀-ICG₁ 29.1 11927.6 ± 1.0 6.91 0.13 51.6 ^(a)Number-averaged molecular weights (M_(n))were determined by GPC using THF as the eluent; ^(b)Repeating unit wascalculated based on integrations of —CH₂—O— groups on PDPA to themethylene groups on PEG using ¹H NMR; ^(c)Determined as ICG fluorescenceemission intensity in 50% human serum.

To assess the breadth of tumor detection, three orthotopic head and necktumors (HN5, FaDu and HCC4034, a tumor xenograft from a patient ofB.D.S), a subcutaneous breast tumor (MDA-MB-231), an intramammaryorthotopic breast tumor (triple negative 4T1), a peritoneal metastasismodel from HCT116 colorectal cancer cells, a patient derived xenograftof kidney cancer, and an orthotopic brain tumor from U87 glioma cellswere imaged. All the tumors were established in NOD-SCID mice except 4T1tumors in immunocompetent BalB/C mice. Bright fluorescent illuminationwas observed across all the tumor types (FIG. 48). Ex vivo imagingrevealed high contrast ratios of tumor over muscle (20-50 fold) withhigh cancer specificity (FIGS. 49A & 49B). Using HN5 tumor model, thecompatibility of PINS imaging with multiple clinical cameras wasdemonstrated (FIGS. 50A-50F).

Using the SPY camera, real-time tumor acidosis guided surgery (TAGS) inmice bearing HN5 head and neck or 4T1 breast cancers was performed. PINS(2.5 mg/kg) was injected intravenously 12-24 h before surgery. In arepresentative operation in HN5 tumor-bearing mice, after resection ofthe primary tumor, the residual tumor was clearly visible by the SPYcamera (middle left panel in FIG. 51A) but not under white light (topleft panel). To investigate the accuracy of margin delineation,non-survival surgery in 9 mice bearing HN5 head and neck tumors wereanalyzed using a double blind protocol. The surgeon resected the tumorsunder PINS illumination and marked the tissue specimen (2-3 mm in size)as either primary tumor, tumor margin or negative muscle tissue based onfluorescence. The specimens were then frozen sectioned and stained withH&E. Histological evaluation was performed independently by a clinicalpathologist (FIG. 52). Using histology as the gold standard, PINSfluorescent assessment had a 95% confidence of detection accuracybetween 89.5% and 100% (n=27). Long-term survival surgery outcomes showimproved loco-regional control and overall survival with TAGS over whitelight surgery (WLS), debulking surgery and untreated controls (FIG.51B). Debulking surgery with macroscopically positive margins typicallyprovides no survival benefit for head and neck cancer and served as acontrol for the adequacy of WLS. WLS was superior to the debulking anduntreated controls (P<0.0001) which showed equivalent survival,indicating good unbiased technique. TAGS led to the best outcome, with13 out of 18 animals (72%) showing cures 150 days post-operatively(P<0.0001 vs. WLS, FIG. 51B).

To mimic clinical scenarios where identifying occult cancerous nodulesmay take precedence over tumor margins, small orthotopic breast tumorswere established in immunocompetent female BalB/C mice. 5×10⁴ triplenegative 4T1 breast cancer cells were injected in the inguinal mammarypad. With an estimated doubling time of 24 h, the nodule size represents<1 million 4T1 cells in the foci on day 4. PINS under SPY camera wasable to identify the 4T1 foci, which was confirmed by histology (FIGS.53A-53C). Tumor could not be detected with visual inspection orpalpation. For the white light control, the tumor was allowed to grow to˜25 mm³ to be visible, and carefully resected the primary tumor andsurrounding margin. TAGS resulted in superior visualization, improvingsurvival after resection over the untreated control and WLS (P<0.05,FIG. 53D), demonstrating superb imaging sensitivity with PINS.

Tumor response to small molecular inhibitors targeting different tumoracidosis pathways was evaluated by PINS (FIGS. 54A-54C). Four inhibitorswere selected: acetazolamide for carbonic anhydrase IX (CAIX),V) (Neri &Supuran, 2011) α-cyano-4-hydroxycinnamate (CHC) for monocarboxylatetransporter (MCT) (Sonveaux et al., 2008), cariporide for sodium protonex changer 1 (NHE1) (Cardone et al., 2005) and pantoprazole as a protonpump inhibitor (PPI) (Vishvakarma & Singh, 2011). The PINS was injectedintravenously to BalB/C mice bearing 4T1 tumors following inhibitoradministration. NIR imaging 24 h after PINS injection showed drasticinhibition (74.2%) by CAIX inhibitor acetazolamide over PBS control.Moderate inhibition (29.3%) by MCT inhibitor CHC was also observed. Nosignificant inhibition was noticed by cariporide or pantoprazole. ThePINS response is consistent with the previously reported antitumorefficacy of CAIX inhibitors in 4T1 tumors (Lou et al., 2011 andPacchiano et al., 2011). Compared to ¹H/³¹P¹⁹ or hyperpolarization ¹³CMRI methods (Gallagher et al., 2008), PINS imaging offers a simple andconvenient reporter assay for the mechanistic investigation of tumoracidosis and development of drugs that target dysregulated pH of solidcancers (Neri & Supuran, 2011 and Parks et al., 2013).

Safety evaluation of the PINS in immunocompetent C57BL/6 mice showedtemporary body weight loss at high dose (FIG. 55A and Tables 8 & 9). Themaximum tolerated dose is at 250 mg/kg, 100-fold higher than the imagingdose. Mice were sacrificed on day 1, 7 and 28 at 200 and 250 mg/kg.Liver and kidney functions were measured (FIGS. 55B-55D). Liver enzymelevels (ALT and GOT) increased on day 1 after PINS injection andreturned to normal after 7 days. Histology analysis (FIG. 56) showedmicrosteatosis in the liver in the 250 mg/kg group at day 1 and returnedto normal by 28 days. Other major organs (e.g., kidney, heart, spleen,brain) are normal.

TABLE 8 Log-rank p-values for pairwise treatment comparisons amongdifferent groups in survival surgery. Debulking White light TAGS Headand Control 0.347 <0.0001 <0.0001 neck surgery Debulking — <0.0001<0.0001 White light — — <0.0001 Breast Control —  0.0501 <0.0001 surgeryWhite light — — 0.0116

TABLE 9 Tolerability and survival of C57BL/6 mice following bolusinjection of PINS. 7 Days Morbidity reaction type and degree^(a)mortality: Lack of Lack of Dose n death/total n Reduced mobilityappetite (mg/kg) (% deaths) feces (<6 h) (<6 h) 150 0/5 (0%) − recoveredsoon recovered soon 200 0/10 (0%) + + + 250 0/15 (0%) ++ ++ ++ 300 4/5(80%) +++ +++ +++ ^(a)Reaction degree was recorded as: − no reaction; +mild reaction; ++ intermediate reaction; +++ strong reaction.

2. Materials and Methods

Characterization of Monomer and Polymer of PINS.

Syntheses of 2-(ethylpropylamino)ethyl methacrylate (EPA-MA) andpoly(ethylene glycol)-b-poly(ethylpropylaminoethyl methacrylate)copolymers (PEG-b-(PEPA)) were described in the method section above.Below are the chemical characterizations of the monomer and copolymer:

2-(Ethylpropylamino) Ethyl Methacrylate (EPA-MA)

¹H NMR (TMS, CDCl₃, ppm): 6.10 (s, 1H, CHH═C(CH₃)—), 5.54 (s, 1H,CHH═C(CH₃)—), 4.20 (t, 2H, —OCH₂CH₂N—), 2.75 (t, 2H, —OCH₂CH₂N—), 2.58(q, 2H, —N(CH₂CH₂CH₃)(CH₂CH₃)), 2.44 (m, 2H, —N(CH₂CH₂CH₃)(CH₂CH₃)),1.94 (s, 3H, CH₂═C(CH₃)—), 1.45 (m, 2H, N(CH₂CH₂CH₃)(CH₂CH₃)), 1.02 (t,3H, —N(CH₂CH₂CH₃)(CH₂CH₃)), 0.87 (t, 3H, —N(CH₂CH₂CH₃)(CH₂CH₃)). ¹³CNMR(CDCl₃, ppm): 167.42, 136.36, 125.35, 63.20, 56.31, 51.51, 48.32, 20.54,18.33, 12.09, 11.82. [M+H]+: 200.2 (calculated 200.3).

Poly(ethylene glycol)-b-poly(ethylpropylaminoethyl methacrylate)(PEO-b-P(EPA)₁₀₀)

¹H NMR (TMS, CDCl₃, ppm): 3.99 (b, 204H, —COOCH2-), 3.83-3.45 (m, 450H,—CH2CH2O—), 3.38 (s, 3H, CH3O—), 2.68 (b, 204H, —OCH2CH2N), 2.55 (b,204H, N(CH₂CH₂CH₃)(CH2CH3)), 2.41 (b, 204H, —N(CH₂CH₂CH₃)(CH₂CH₃)),1.78-1.90 (m, 270H, CCH3C & C(CH3)₂), 1.45 (m, 204H,—N(CH2CH2CH3)(CH₂CH₃)), 1.02 (b, 306, —N(CH₂CH₂CH₃)(CH₂CH₃)), 0.88 (b,306H, —N(CH₂CH₂CH₃)(CH₂CH₃)). ¹³CNMR (CDCl₃, ppm): 177.73, 177.33,176.61, 70.58, 63.26, 63.13, 56.21, 51.09, 45.05, 44.70, 38.69, 31.92,30.33, 29.69, 29.36, 28.90, 23.72, 22.98, 22.69, 20.62, 16.53, 14.13,12.18, 11.91.

Fluorescence Activation of PINS.

Fluorescence intensity of PINS in different pH buffer solutions wasmeasured on a Hitachi fluorimeter (F-7500 model). For each PINScomposition, a stock solution in MilliQ water at the concentration of2.5 mg/mL was prepared. The stock solution was then diluted with either80 mM phosphate-buffered saline (PBS) buffer with different pH values or50% human serum in 80 mM PBS buffer with different pH values. The finalmicelle concentration was controlled at 0.05 mg/mL in PBS or 0.025 mg/mLin 50% human serum. The nanoprobe solution was excited at 780 nm and theemission spectra were collected from 800 nm to 900 nm. The emissionintensity at 815 nm in PBS and 830 nm in 50% human serum was used toquantify the pH-response of the nanoprobes. Fluorescent images of PINSsolution (0.05 mg/mL) in test tubes at different pH values were taken bya SPY Elite® imaging system.

Shelf-Life Study.

Freshly prepared nanoprobe aqueous solution (5 mg/mL) was mixed withequal volume of 20% sucrose aqueous solution to generate 2.5 mg/mL stocksolution in 10% sucrose. The stock solution was divided and sealed inseveral test tubes and frozen in a −20° C. freezer. Samples were thawedat designated time point to test the fluorescence activation in PBS or50% human serum as described above.

Cell Culture.

The cancer cell lines used for in vivo tumor models include HN5, FaDu,HCC4034 human head and neck cancers, MDA-MB-231 and 4T1 breast cancers,U87 glioma, and HCT116 colorectal cancer cells. HN5 and FaDu cell lineswere obtained from Michael Story's lab; HCC4034 was established by JohnMinna's lab from a resected tumor of a head and neck patient of Dr.Baran Sumer; MD-MBA-231, 4T1 and HCT116 were obtained from DavidBoothman lab; U87 was obtained from Dawen Zhao lab. All cells lines weretested for mycoplasma contamination before use. Negative status forcontamination was verified by Mycoplasma Detection Kit from Biotool.Cells were cultured in DMEM with 10% fetal bovine serum and antibiotics.

Animal Models.

Animal protocols related to this study were reviewed and approved by theInstitutional Animal Care and Use Committee. Female NOD-SCID mice (6-8weeks) were purchased from UT Southwestern Medical Center Breeding Core.For orthotopic head and neck tumors, HN5, FaDu or HCC4034 cells (2×10⁶per mouse) were injected into the submental triangle area. One weekafter inoculation, animals with tumor size 100-200 mm³ were used forimaging studies. Subcutaneous breast tumor model was established byinjecting MDA-MB-231 (2×10⁶ per mouse) cells on the right flank.Peritoneal metastasis was established by intraperitoneal injection ofHCT-116 (2×10⁶ per mouse) cells followed by gentle massage on theabdomen. Orthotopic U87 glioma bearing mice were established byintracranial injection of U87 cells. Mice bearing XP296 patient-derivedkidney xenograft were provided by the James Brugarolas lab. FemaleBalB/C mice (6-8 weeks) were purchased from UT Southwestern MedicalCenter Breeding Core. Orthotopic breast tumor model was established inBalB/C mice by injection of 4T1 (5×10⁴ per mouse) cells into the rightthoracic mammary glands.

Dose-Response Study.

HN5-tumor-bearing mice (3 for each group) were injected with 1.0, 2.5 or5.0 mg/kg PINS isotonic solution. The control group was injected with0.08 mg/kg free ICG dye (equivalent to the dye content in 2.5 mg/kgPINS). At designated time point, mice were anesthetized with 2.5%isofluorane and imaged with SPY Elite®. Fluorescence intensity wasmeasured by Image J. Contrast to noise ratios (CNR) were calculated bythe following equation:

${C\; N\; R} = \frac{{F\; {I({Tumor})}} - {F\; {I\left( {{Normal}\mspace{14mu} {Tissue}} \right)}}}{s.d.\; \left( {{Normal}\mspace{14mu} {Tissue}} \right)}$

FI(Tumors) and FI(Normal Tissue) are the fluorescence intensities of thetumor and normal tissues, respectively. The background noise wasmeasured as the standard deviation of the normal tissue fluorescence.

In Vivo and Ex Vivo Fluorescence Imaging.

Nanoprobes (2.5 mg/kg for all tumor models except 3.0 mg/kg for U87 andXP296) were administered intravenously via the tail veins oftumor-bearing mice. After 24 h, the animals were imaged by the SPYElite® clinical camera. For ex vivo imaging, tumors and main organs wereharvested and imaged. Fluorescence intensities of the tumors and organswere normalized to the muscle tissue of comparable size.

Example 7: Use of Micelles to Evaluate Endocytic Organelles and theirUse in Signaling and Proliferation

1. Preparation of the pH Responsive Systems

In order to evaluate the physiological roles of organelles, a series ofamphiphilic block copolymers PEO-b-P(R₁-r-R₂), where PEO ispoly(ethylene oxide) and P(R₁-r-R₂) is an ionizable random copolymerblock were synthesized (FIG. 57A and FIG. 58). The molecular compositionof each copolymer is shown in Table 10. At high pH (e.g., 7.4 inphosphate-buffered saline), these copolymers self-assemble intocore-shell micelle structures (diameter 30-60 nm, surface electrostaticpotential −2 to 0 mV, Table 10 and FIG. 59). At pH below the apparentpK_(a) of each copolymer, micelles dissociate into unimers due to theprotonation of tertiary amines. The previous studies exploited the sharppH-dependent micelle transitions for the development of a series oftunable, ultra-pH sensitive fluorescence sensors (Ma, et al., 2014).

TABLE 10 Chemical compositions and physical properties of UPSnanoparticles. Com- position^(a) D^(h) (nm)^(b) PDI^(b) ξ (mV)^(c)pK_(a) ^(d) pH_(t) ^(e) UPS_(4.4) P(D5A₈₀) 47.5 ± 3.0 0.13 ± 0.01 −1.1 ±0.2 4.35 4.39 UPS_(4.7) P(DBA₂₈- 62.4 ± 2.9 0.08 ± 0.01 −0.5 ± 0.1 4.654.71 D5A₅₂) UPS_(5.0) P(DBA₅₆- 54.6 ± 1.2 0.10 ± 0.01 −1.3 ± 0.4 4.935.02 D5A₂₄) UPS_(5.3) P(DBA₈₀) 42.3 ± 2.6 0.12 ± 0.02 −0.7 ± 0.1 5.315.32 UPS_(5.6) P(DPA₃₀- 49.8 ± 2.6 0.11 ± 0.01 −2.1 ± 0.4 5.58 5.61DBA₅₀) UPS_(5.9) P(DPA₆₀- 49.2 ± 1.3 0.11 ± 0.01 −0.9 ± 0.1 5.89 5.91DBA₂₀) UPS_(6.2) P(DPA₈₀) 44.3 ± 1.2 0.10 ± 0.01 −1.6 ± 1.8 6.19 6.22UPS_(6.5) P(DEA₂₁- 42.0 ± 1.3 0.12 ± 0.02 −0.9 ± 0.6 6.45 6.50 DPA₇₉)UPS_(6.8) P(DEA₃₉- 35.2 ± 1.3 0.11 ± 0.01 −1.4 ± 0.6 6.77 6.79 DPA₆₁)UPS_(7.1) P(DEA₅₈- 32.7 ± 1.3 0.13 ± 0.01 −0.9 ± 1.1 7.05 7.08 DPA₄₂)^(a)Only the composition of the PR segment is shown. The subscriptsindicate the number of repeating unit for each monomer. ^(b)Thehydrodynamic diameter (D_(h)) and polydispersity index (PDI) wereanalyzed by dynamic light scattering analysis. ^(c)Surface electrostaticpotential (ξ) of the UPS nanoparticles was analyzed by the Zeta Sizer.^(d)The apparent pK_(a) values for UPS nanoparticles were measured by pHtitration experiments in the presence of 150 mM NaCl. ^(e)The transitionpH (pH_(t)) was measured from Cy5-conjugated UPS nanoprobes based onfluorescence intensity.

Herein are described UPS nanoparticles that have exquisite pH-tunablebuffer capacity at a narrow pH interval in a broad range of pH (4.0 to7.4). FIG. 57B shows the pH titration curves of three exemplaryUPS_(4.4), UPS_(5.3), and UPS_(6.2) nanoparticles (each subscriptindicates the pK_(a) of the corresponding copolymer, Table 10) in thepresence of 150 mM NaCl. Results showed that UPS_(4.4), UPS_(5.3), andUPS_(6.2) (2 mg/mL) were able to buffer the pH at their apparent pK_(a)at 4.4, 5.3 and 6.2, respectively, when HCl (0.4 M) was added into thepolymer solution. In contrast, chloroquine (CQ), a widely used smallmolecular base in biological studies, showed a broad pH response in therange of pH 6 to 9 (pK_(a)=8.3), as well as polyethyleneimines as abroad pH buffer (Suh et al., 1994). Determination of buffer capacityfrom the pH titration curves (FIG. 57C and FIG. 60) showed exquisitebuffer strengths at specific pH in the range of pH 4.0-7.4. Morespecifically, the maximal values for UPS_(4.4), UPS_(5.6), and UPS_(7.1)nanoparticles were 1.4, 1.5 and 1.6 mmol HCl per 40 mg of nanoparticle,which are 339-, 75- and 30-fold higher than CQ at pH 4.4, 5.6 and 7.1,respectively (FIG. 57C). This collection of UPS nanoparticles provides aunique set of pH-specific “proton sponges” for the functional range oforganelle pH from early endosomes (E.E., 6.0-6.5) (Weisz, 2003) to lateendosomes (L.E., 5.0-5.5) (Weisz, 2003) to lysosomes (4.0-4.5) (Casey etal., 2010).

2. pH Buffering Capacity and Proton Pumping Rates

For simultaneous imaging and buffering studies, a new nanoparticledesign with a dual fluorescence reporter was established: an “always ON”reporter to track intracellular nanoparticle distribution regardless ofthe pH environment, and a pH-activatable reporter (OFF at extracellularmedium pH 7.4 and ON at specific organelle pH post endocytosis). Initialattempts at conjugating a dye (e.g., Cy3.5) on the terminal end of PEOsucceeded in an always ON signal, however, the resulting nanoparticleswere unstable as a result of dye binding to serum proteins. To overcomethis limitation, a heteroFRET design using a pair of fluorophores thatwere introduced in the core of micelles was employed. As an example, aFRET pair (e.g., BODIPY and Cy3.5 as donor and acceptor, respectively)was conjugated to the PR segment of the UPS6.2 copolymer. Mixing of thetwo dye-conjugated copolymers (optimal molar ratio ofdonor/acceptor=2:1) within the same micelle core allowed theheteroFRET-induced fluorescence quenching of donor dye (e.g., BODIPY) inthe micelle state (pH>pK_(a)), but fluorescence recovery in the unimerstate after micelle disassembly at lower pH (FIG. 61A upper panel). Togenerate the “always ON” signal, the weight fraction of Cy3.5-conjugatedcopolymer in the micelles was kept low (e.g., 40%) to avoidhomoFRET-induced fluorescence quenching for the acceptor dye in themicelle state (Zhou, et al., 2012) (FIG. 61B). The resulting UPSnanoparticle showed constant fluorescence intensity in the Cy3.5 channelacross a broad pH range, while achieving ultra-pH sensitive activationat specific pH for BODIPY signal (FIG. 61C). Since both fluorophoreswere embedded within the micelle core, the resulting UPS nanoparticleswere stable and free from protein bindings.

UPS_(6.2) and UPS_(5.3) were chosen for imaging and buffering studysince their apparent pK_(a)'s correspond to early endosomes to lateendosomes and to lysosomes transitions, respectively (Weisz, 2003). HeLacells were incubated with an increasing dose (100, 400 and 1,000 μg/mL)of UPS_(6.2) or UPS_(5.3) for 5 min at 37° C. to allow particle uptakevia endocytosis (Conner & Schmid, 2003), then washed with fresh medium(10% FBS in DMEM). At 100 μg/mL, half maximal UPS_(6.2) activation(BODIPY channel) was observed by 30 min and half maximal UPS_(5.3)activation by 60 min (FIGS. 62A & 62B for UPS_(6.2); FIGS. 63A & 63B forUPS_(5.3)). In contrast, at 1,000 μg/mL, activation of BODIPY signal wasdelayed by at least 60 min despite clear indication of particle uptakein the HeLa cells by the Cy3.5 signal (FIGS. 62A & 62B and FIGS. 63A &63B). In situ quantitation of the endosomal pH with Lysosensor showeddose-dependent sustained pH plateaus at pH 6.2 and 5.3 upon exposure ofcells to 400 and 1,000 μg/ml UPS_(6.2) (FIG. 62C) and UPS_(5.3) (FIG.63C), respectively. For either nanoparticle, 100 μg/ml dose wasinsufficient to delay organelle acidification.

To further quantify the acidification rates, the number of micellenanoparticles per HeLa cell was measured based on the fluorescenceintensity of internalized UPS divided by the cell number (see methodsbelow). Data shows an increasing number of nanoparticles at higherincubation doses (Table 11). Based on the number of amino groups permicelle (64,000) and an average of 200 endosomes/lysosomes per cell(Holtzman, 1989), the acidification rate was calculated as approximately150-210 protons per second for each organelle. This result is consistentwith calculations based on 2 protons per ATP hydrolyzed per V-ATPase(Deamer et al., 1999), 3 ATP molecules consumed per rotation (Cross &Muller, 2004), 2.4 revolutions per second (Imamura et al., 2003) and anaverage of 20 V-ATPases per organelle (Imamura et al., 2003).

TABLE 11 Quantification of acidification rates of endocytic organellesby the UPS nanoparticles. [UPS]_(med) D_(h)/ξ No. UPS/cell Plateau pHt_(p) Proton rate (μg/mL) (nm/mV)^(a) (×10²)^(b) (mean ± SD) (min)^(c)(×10²/sec)^(d) UPS_(6.2) 100 42.3 ± 2.6/ 4.7 n.d. n.d. N/A 400 −0.7 ±0.1 17 6.2 ± 0.1 51 1.8 1,000 24 6.2 ± 0.1 84 1.5 UPS_(5.3) 100 44.3 ±1.2/ 4.6 n.d. n.d. N/A 400 −1.6 ± 1.8 17 5.4 ± 0.1 43 2.1 1,000 24 5.3 ±0.1 68 1.9 ^(a)Hydrodynamic diameter (D_(h)) and zeta potential (ξ) weremeasured in the PBS buffer at pH 7.4. ^(b)Calculated based on 800copolymer chains per micelle. ^(c)t_(p) is measured as the time intervalwhere the pH was buffered at the plateau value. ^(d)The rate of protonpumping into each endocytic organelle.

3. pH Thresholds for Two Different Modes of mrTORC1 Activation

The consequences of UPS buffering of luminal pH on endosome protein coatmaturation and endo/lysosome-dependent signal transduction wereexamined. For this purpose, UPS nanoparticles that discretely report andbuffer at pH 6.2, 5.3, 5.0, 4.7 and 4.4 were selected. This range coversestablished luminal pH values in early endosomes, late endosomes andlysosomes. A discriminating feature of early endosome biogenesis isrecruitment of the Rab5 GTPase (Huotari, & Helenius, 2011), whichcorresponds to a luminal pH range of 6.0-6.5 (Weisz, 2003). Fully maturelysosomes are LAMP2 positive with a luminal pH range of 4.0-4.5 (Caseyet al., 2010). To enable quantitation of colocalization of UPS positiveendosomes with endosomal maturation markers, Cy5-encoded UPS_(6.2) andUPS_(4.4) were developed with a low dye/polymer ratio that allowed fordetectable fluorescence in the micelle state (Wang et al., 2014) (FIG.64A). Within 15 min at a concentration of 1,000 μg/mL, over 60% ofUPS_(6.2) and UPS_(4.4) positive endosomes were also Rab5 positive(FIGS. 64A & 64D). UPS_(4.4) positive endosomes further transitioned toa Rab5 negative/LAMP2 positive maturation state within 60 min (FIGS. 64Band 64E-64F). Notably, UPS_(6.2) positive endosomes also became LAMP2positive in a similar timeframe despite inhibition of the luminalacidification that normally accompanies this transition (FIGS. 64B &64D). However, UPS_(6.2) delayed release of Rab5, resulting in transientaccumulation of anomalous Rab5/LAMP2 positive endosomes at 60 min (FIGS.64B & 64F). These observations indicate the presence of a regulatorymechanism that recruits LAMP2 to nascent endolysomes independent of theluminal pH and the presence of a luminal pH-sensitive Rab5 releasemechanism.

To examine the consequence of luminal pH clamping on endo/lysosomebiology, a key regulatory system was evaluated which has recentlyreported to be linked to lysosome biogenesis-namely nutrient dependentactivation of cell growth via mammalian target of rapamycin complex 1(mTORC1). In mammalian cells, mTORC1 localizes to endo/lysosomalmembranes in response to internalized free amino acids (Sancak et al.,2010). Furthermore, the physical interactions between the V-ATPase andRag GTPases on endo/lysosomal membranes are needed for mTORC1 activationin response to nutrient availability (Zoncu et al., 2011). To evaluateamino-acid induced mTORC1 activation, two quantitative reporters ofmTORC1 pathway activation were employed: phosphorylation/activation ofthe mTORC1 substrate p70S6 kinase (p70S6K) and nuclear/cytoplasmicdistribution of the mTORC1 substrate TFEB.

Incubation of HeLa cells for 2 h in a nutrient-free balanced saltsolution (EBSS) was sufficient to inhibit mTORC1 activity as indicatedby reduced accumulation of activation site phosphorylation on bothp70S6K and its substrate S6. Addition of essential amino acids wassufficient to induce pathway activation within 5 min (FIG. 65A and FIGS.66A & 66B). Pretreatment with 1,000 μg/ml of UPS_(4.7), or UPS_(4.4) hadlittle to no effect on the mTORC1 response to free amino acids. Incontrast, pretreatment with 1,000 μg/ml UPS_(6.2), UPS_(5.3) andUPS_(5.0) both delayed and significantly suppressed the mTORC1 pathwayresponse to free amino acids (FIGS. 65A & 65B and FIGS. 66A & 66B). Theselective UPS inhibition of the mTORC1 pathway response was mirrored byTFEB nuclear/cytoplasm distribution. Phosphorylation of thistranscription factor by mTORC1 results in nuclear exclusion, therebyinhibiting the TFEB transcriptional program in nutrient repleteconditions (Pena-Llopis et al., 2011, Settembre et al., 2012 andRoczniak-Ferguson et al., 2012). In Hela cells, with stable expressionof GFP-tagged TFEB, pretreatment with UPS_(6.2), UPS_(5.3) and UPS_(5.0)inhibited redistribution of TFEB to the cytoplasm upon addition of freeamino acids. In contrast, in cells pretreated with UPS_(4.7) andUPS_(4.4), TFEB redistribution proceeded normally (FIGS. 65C & 65D).

The above data suggest acidification of endosomes below a threshold ofpH 5 is necessary for free amino acid-induced activation of mTORC1.Similar experiments were performed employing bovine serum albumin (BSA)as a macromolecular nutrient source rather than free amino acids.Similar to free amino acids, BSA exposure was sufficient to reactivatemTORC1 following nutrient starvation (FIGS. 67A-67D). However, incontrast to free amino acids, UPS_(4.4) delayed mTORC1 activation inresponse to BSA (FIGS. 67A & 67D). Given that cells treated withUPS_(4.4) responded normally to free amino acids, the delayed responsewas surmised to BSA is the consequence of inhibition of the proteolysisof BSA by acid hydrolases in the lysosome. Consistent with this,significant inhibition of cathepsin B activity in the presence ofUPS_(4.4) was found (FIG. 66C). Together, these observations indicatethat distinct lysosomal pH thresholds are required for acid hydrolaseactivity versus free amino acid sensing (FIG. 65E).

4. Modulating Cellular Metabolite Pools by Buffering Lysosomal pH

Lysosomes recycle intracellular macromolecules and debris to producemetabolic intermediates deployed for energy production or forconstruction of new cellular components in response to the nutrientstatus of the cellular environment (Settembre et al., 2013). Abnormalaccumulation of large molecules, including lipids and glycoproteins inlysosomes are associated with metabolic disorders. To broadly assessalterations associated with highly selective perturbation of lysosomalacidification, accumulation of small metabolites in cells was quantifiedby loaded with UPS_(4.4) under nutrient starved versus nutrient repletegrowth conditions. Following a 12 h exposure to 0, 200, and 400 g/ml ofUPS_(4.4), HeLa cells were lysed and intracellular metabolites werequantified using liquid chromatography-triple quadrupole massspectrometry (LC/MS/MS). Sixty-eight metabolites were quantifiable from3×10⁶ HeLa cells, revealing a number of dose-dependent andnutrient-dependent consequences of pH arrest at 4.4 in lysosomes (FIG.68A). Under nutrient replete conditions, as the dose of UPS_(4.4)increased, the relative abundance of most metabolites also increasedwhen normalized to cellular protein content. This included most aminoacids (FIG. 68B upper panel), consistent with an inhibition of theanabolic signals required to use them for protein synthesis and/ordefects in lysosomal export of amino acids. In nutrient deprivedconditions, UPS_(4.4) enhanced the relative abundance of nucleotides andtheir precursors (e.g., bottom cluster in FIG. 68A) and massivelysuppressed the second messenger cAMP. The loss of many essential aminoacids including lysine, valine, methionine, and arginine was alsoobserved, consistent with the inhibition of starvation-inducedcatabolism of macromolecules like albumin (FIG. 68B lower panel). Theseresults suggest mechanistic connections between organelle acidificationand metabolite pools, and fortify the hypothesis that proper lysosomalacidity is required for homeostasis of numerous metabolic pathways,either in the presence or absence of nutrients (FIG. 68C).

5. Effects of NSCLC Cells to Endo/Lysosomal pH Arrest

The inventors recently described a selective metabolic vulnerability innon-small cell lung cancer (NSCLC) cells, whereby co-occurring mutationsin the KRAS oncogene and LKB1 tumor suppressor result in cellularaddiction to lysosomal catabolism for maintenance of mitochondrialhealth (Kim et al., 2013). Genetic or chemical inhibition of V-ATPaseactivity was sufficient to selectively induce programmed cell death inthis oncogenic background. This was proposed to be a direct consequenceof inhibition of a lysosome-dependent supply of TCA cycle substrates forATP production. The UPS library afforded an opportunity to directly testthis hypothesis in the absence of confounders associated with thepleiotropic contributions of V-ATPases to cytosolic pH and mTORC1/AMPKprotein complexes in cancer cells (Zoncu et al., 2011 Zhang et al.,2014). As a model system, normal (HBEC30KT) and tumor-derived (HCC4017)cell lines from the same patient were employed together with an isogenicprogression series in which the KRAS and LKB1 lesions were artificiallyintroduced into the normal cell background (FIG. 69A) (Ramirez et al.,2004). A comparison of cell number and morphology between HCC4017 andHBEC30KT treated with UPS_(6.2), UPS_(5.3) and UPS_(4.4) at high doserevealed highly selective toxicity of these UPS nanoparticles to HCC4017(FIG. 69B). The expression of oncogenic KRAS together with inhibition ofLKB1 was sufficient to induce sensitivity of bronchial epithelial cellsto UPS-induced programmed cell death (FIGS. 69C-69E). Importantly, thisphenotype was rescued in both the tumor-derived cells (FIG. 69G) and thegenetically engineered cells (FIG. 69G) upon addition of cell permeableanalogs of TCA cycle substrates (methyl pyruvate and α-ketoglutarate).Thus selective vulnerability of KRAS/LKB1 co-mutant NSCLC cells tolysosomal function arises from addiction to catabolism of extracellularmacromolecules. Moreover, the higher cytotoxicity by UPS_(6.2) overUPS_(4.4) indicates mTORC1 inhibition further contributed to thelethality in these cells.

6. Discussion

Luminal acidification is a hallmark of maturation of endocyticorganelles in mammalian cells conferring distinctive cellular functionssuch as receptor recycling, organelle trafficking and protein/lipidcatabolism at different stages (Maxfield & McGraw, 2004 and Yeung etal., 2006). Existing tools or reagents (e.g., chloroquine, NH₄Cl,bafilomycin A1) are cell permeable and block a broad range of pHactivities. Consequently, biological interrogations on endosome/lysosomefunctions using these agents may suffer from compounded, non-specific pHeffect as well as contributions from perturbation of other acidicorganelles (such as the Golgi). In contrast, current UPS nanoparticlesenter cells exclusively through endocytosis; furthermore, they allow forrobust and fine-scale buffering of luminal pH at operator-predeterminedthresholds along the endocytic pathway. The exquisite pH-specific buffereffect, together with previously reported ultra-pH sensitivefluorescence response (Zhou et al., 2011 and Zhou et al., 2012), areunique nanoscale property in self-assembled systems, where hydrophobicmicellization (phase transition) dramatically sharpens the pH transitionleading to cooperative protonation of tertiary amines. As a result, theUPS nanoparticles yielded a high resolution buffer effect within 0.3 pHunit. The buffered pH range (centered around apparent pK_(a)) of the UPSplatform can be fine-tuned by the hydrophobicity of the PR segment,unlike small molecular pH buffers/sensors that are mostly controlled byelectron withdrawing/donating substituents (Urano, et al., 2008). Theunique pH-specific, tunable “proton sponge” effect is distinct fromother low resolution polybase buffers (e.g., polyethyleneimines, FIG.57C). To further achieve simultaneous imaging and buffering capability,an always-ON/OFF-ON composition was constructed employing a heteroFRETstrategy. This nanoparticle design permitted the first measurement ofacidification rates of the endocytic organelles (150-210 protons persecond) in the HeLa cells, which is in the same order of magnitude withestimations (240-310) based on literature data.

Detailed evaluation of the UPS library illustrated how perturbation ofluminal pH of endocytic organelles impacted multiple cell physiologicalprocesses, which contributes to the understanding of endosome biologyand bio-nano interactions. More specifically, the “perturb and report”characteristics of the library allowed for time-resolved quantitation ofendosome maturation, and uncovered previously unappreciated consequencesof luminal pH on endosomal coat protein exchange. Notably, therecruitment of the “mature” lysosome marker, LAMP2, was found to occurindependently of luminal acidification. On the other hand, release ofthe early endosome marker Rab5 is delayed by luminal alkalization,resulting in the de novo accumulation of Rab5/LAMP2 positive endosomes.This indicates the presence of currently undescribed, but explorable,pH-sensitive and pH-insensitive mechanisms governing endosome/lysosomebiogenesis. The ability to fine-tune UPS buffering capacity also alloweddiscrimination of distinct pH thresholds required for free amino acidversus albumin dependent activation of mTORC1 pathway. Without wishingto be bound by any theory, it is believed that the acidification to pH5.0 or below is required to release free amino acids for “inside-out”communication with V-ATPase protein complexes, or for induction ofconformational changes in V-ATPase during amino acid sensing (Zoncu etal., 2011). Similarly, acidification to pH 4.4 or below is used inalbumin dependent activation of mTORC1, most likely due to the need forhydrolase activation and subsequent protein catabolism. The scalabilityof UPS synthesis enabled broad-spectrum quantitation of the cellularmetabolite milieu upon inhibition of lysosomal consumption ofextracellular macromolecules. The exclusive uptake of UPS withinendocytic organelles afforded the opportunity to specifically evaluatethe participation of endosomal/lysosomal pH in growth regulatorysignaling pathways and cell metabolism.

7. Methods

1. Chemicals

The Cy5-NHS, BODIPY-NHS and Cy3.5-NHS esters were purchased fromLumiprobe Corp. (FL, USA). Monomers 2-(diethylamino) ethyl methacrylate(DEA-MA) and 2-aminoethyl methacrylate (AMA) were purchased fromPolyscience Company. Monomers 2-(dibutylamino) ethyl methacrylate(DBA-MA) (Zhou et al., 2011), 2-(dipropylamino) ethyl methacrylate(DPA-MA) and 2-(dipentylamino) ethyl methacrylate (D5A-MA) (Li et al.,2014) were prepared according to the method described in the inventor'sprevious work, as well as the PEO macroinitiator (MeO-PEO₁₁₄-Br)¹.N,N,N′,N″,N′″-Pentamethyldiethylenetriamine (PMDETA) was purchased fromSigma-Aldrich. Amicon ultra-15 centrifugal filter tubes (MWCO=100 K)were obtained from Millipore (MA). Other reagents and organic solventswere analytical grade from Sigma-Aldrich or Fisher Scientific Inc.

2. Cells, Culture Media and Biological Reagents

The NSCLC cell line HCC4017 and its matched normal bronchial epithelialcell line HBEC30KT were developed from the same patient. The generationof these cell lines and the corresponding HBEC30KT oncogenic progressionseries was as previously reported (Ramirez, et al., 2004). HCC4017 andall HBEC30-derived cell lines were cultured in ACL4 medium (RPMI 1640supplemented with 0.02 mg/ml insulin, 0.01 mg/ml transferrin, 25 nMsodium selenite, 50 nM hydrocortisone, 10 mM HEPES, 1 ng/ml EGF, 0.01 mMethanolamine, 0.01 mM O-phosphorylethanolamine, 0.1 nM triiodothyronine,2 mg/ml BSA, 0.5 mM sodium pyruvate) with 2% fetal bovine serum (FBS,Atlanta Biologicals) and 1% antibiotics (GIBCO). HeLa and GFP-TFEB HeLacells were cultured in DMEM (Invitrogen) with 10% FBS and 1% antibiotics(Invitrogen). Earle's Balanced Salt Solution (EBSS, 10×, Sigma) wasdiluted to 1× with Milli-Q water supplemented with 2.2 g/L sodiumbicarbonate (Sigma). Antibodies were from Cell Signaling (S6K-pT389,S6K, S6-Ribosomal-Protein-pS235/236, S6 Ribsomal Protein, Rab5 and Rab7)and Abcam (LAMP2). Other biological agents include Hoechst 33342(Invitrogen), LysoSensor Yellow/Blue DND 160 (Invitrogen), Magic Red™Cathepsin B Assay Kit (Immunochemistry Technology), Bafilomycin A1(Sigma), Chloroquine (Sigma) and BCA Protein Assay Kit (Thermo).

3. Syntheses of Dye-Conjugated PEO-b-(P(R₁-r-R₂)) Block Copolymers

Aminoethyl methacrylate (AMA) was used for the conjugation of dyes.Three primary amino groups were introduced into each polymer chain bycontrolling the feeding ratio of AMA monomer to the initiator (molarratio=3). After synthesis, PEO-b-(PR-r-AMA) (10 mg) was dissolved in 2mL DMF. Then the Dye-NHS ester (1.5 equivalences for Dye-NHS) was added.After overnight reaction, the copolymers were purified by preparativegel permeation chromatography (PLgel Prep 10 μm 10³Å, 300×25 mm columnby varian, THF as eluent at 5 mL/min) to remove the free dye molecules.The resulting copolymers were lyophilized and kept at −20° C. forstorage. The only difference for the syntheses of block copolymers foralways-ON/OFF-ON UPS nanoparticles is that three AMA groups wereintroduced into a polymer chain for BODIPY conjugation, while one AMAgroup was introduced for Cy3.5 conjugation.

4. Preparation and Characterization of UPS Nanoparticle Micelles

In a typical procedure, 10 mg UPS polymer was dissolved in 500 μL THF(without dye conjugation) or methanol (with dye-conjugation). Foralways-on/OFF-ON UPS nanoparticles, BODIPY-conjugated polymer andCy3.5-conjugated polymer was mixed with the indicated weight ratio (FIG.112) to determine the best combination that yields high ON/OFF ratio inBODIPY channel and stable always-on signal in Cy3.5 channel. Thesolution was added to 10 mL Milli-Q water drop by drop. Four to fivefiltrations through a micro-ultrafiltration system (<100 kDa, AmiconUltra filter units, Millipore) were used to remove the organic solvent.The aqueous solution of UPS nanoprobes was sterilized with a 0.22 μmfilter unit (Millex-GP syringe filter unit, Millipore). Transmissionelectron microscopy (TEM, JEOL 1200 EX model, Tokyo, Japan) was used toexamine micelle size and morphology. Dynamic light scattering (DLS,Malvern Nano-ZS model, He-Ne laser, λ=633 nm) was used to determine thehydrodynamic diameter (D_(h)) of 100 μg/mL micelle PBS solutions. Thepresented data were averaged from five independent measurements. Thezeta-potential was measured using a folded capillary cell (MalvernInstruments, Herrenberg, Germany). The presented data were averaged fromthree independent measurements.

5. Quantitation of Cellular Uptake of UPS Nanoprobes

HeLa cells (1×10⁶ per well) were seeded in 6-well tissue culture dishes.After 12 to 16 h, the cells were exposed to UPS_(6.2)-TMR and/orUPS_(5.3)-TMR for 5 min in serum free DMEM, and then washed three timeswith PBS. Following an additional 2 h incubation in DMEM+10% FBS, theUPS nanoprobes were extracted from cells with methanol. UPS nanoprobemicelles disassociate into unimers in methanol. A Hitachi fluorometer(F-7500 model) was used to determine RFU of the UPS-TMR unimer solutionsat 570 nm. The dose of internalized UPS nanoprobes was calculated fromthe RFU and a standard curve of the UPS-TMR solutions.

6. Measurement of Endo/Lysosomal pH

HeLa cells were plated in 4- or 8-well Nunc™ Lab-Tek™ II ChamberedCoverglass (Thermo Scientific) and allowed to grow for 48 h. The cellswere then loaded with 25 μM LysoSensor Yellow/Blue DND-160 and 1,000μg/mL UPS nanoprobes in serum-free medium at 37° C. for 5 min. The cellswere washed twice and immediately imaged. Imaging was performed using anepifluorescent microscope (Deltavision, Applied Precision) equipped witha digital monochrome Coolsnap HQ2 camera (Roper Scientific, Tucson,Ariz.). Fluorescence images were collected using SoftWoRx v3.4.5(Universal Imaging, Downingtown, Pa.). Data were recorded atexcitation/emission wavelengths of 360/460 nm and 360/520 nm. The singleband pass excitation filter for DAPI (360 nm) is 40 nm, and the bandpass of emission filters for DAPI (460 nm) and FITC (520 nm) is 50 nmand 38 nm, respectively. Cell fluorescence ratios were determined byimage analysis of the stored single wavelength images using ImageJsoftware. For each cell, a region of interest was defined as the punctaein cytosol that emitted fluorescent signals from both UPS nanoprobes andLysoSensor. Fluorescent intensity ratio was calculated for eachintracellular punctate as R=(F₁−B₁)/(F₂−B₂) where F₁ and F₂ are thefluorescence intensities at 360/520 and 360/460 respectively, and B₁ andB₂ are the corresponding background values determined from a region onthe same images that was near the punctae in the cytosol. To calibratethe relationship between R and pH, we used a modified protocolestablished by Diwu et al. (1999). Cells were loaded with LysoSensor andthen permeabilized with 10 μM monensin and 10 μM nigericin. These cellswere treated for 30 min with the equilibration buffers consisting of 5mM NaCl, 115 mM KCl, 1.2 mM MgSO₄, and 25 mM MES (MES buffer) variedbetween pH 4.0 and 7.4. The cells were kept in the buffer until imaging.

7. Colocalization Analysis

Images from the immunofluorescence assay were taken by using spinningdisk confocal microscope (Andor). Z-stack images were used afterdeconvolution in the colocalization analysis. The data was analyzedusing the Coloc module of Imaris 7.7 (Bitplane). The thresholdedMander's coefficient was used as an indicator of the proportion of thecolocalized signal over the total signal (Manders et al., 1993 and Bolte& Cordelieres, 2006).

8. Metabolomic Analysis

HeLa cells were grown in 100 mm dishes until 80% confluent, andseparated into nutrient replete and nutrient deplete groups. The mediumfor cells in the nutrient deplete group was changed to EBSS before beingwashed with saline twice. Then 200 or 400 μg/mL UPS_(4.4) (finalconcentration) or same volume of water (as control, each conditioncontains 6 replicates) was added to both groups and was left forovernight. Following this, cells were washed twice with ice cold saline,then overlaid with 500 μL of cold methanol/water (50/50, v/v). Cellswere transferred to an Eppendorf tube and subjected to three freeze-thawcycles. After vigorous vortexing, the debris was pelleted bycentrifugation at 16,000×g and 4° C. for 15 min. Pellets were used forprotein quantitation (BCA Protein Assay Kit, Thermo). The supernatantwas transferred to a new tube and evaporated to dryness using a SpeedVacconcentrator (Thermo Savant, Holbrook, N.Y.). Metabolites werereconstituted in 100 μL of 0.03% formic acid in analytical-grade water,vortex-mixed and centrifuged to remove debris. Thereafter, thesupernatant was transferred to a HPLC vial for the metabolomics study.

Targeted metabolite profiling was performed using a liquidchromatography-mass spectrometry/mass spectrometry (LC/MS/MS) approach.Separation was achieved on a Phenomenex Synergi Polar-RP HPLC column(150×2 mm, 4 am, 80 Å) using a Nexera Ultra High Performance LiquidChromatograph (UHPLC) system (Shimadzu Corporation, Kyoto, Japan). Themobile phases employed were 0.03% formic acid in water (A) and 0.03%formic acid in acetonitrile (B). The gradient program was as follows:0-3 min, 100% A; 3-15 min, 100%-0% A; 15-21 min, 0% A; 21-21.1 min,0%-100% A; 21.1-30 min, 100% A. The column was maintained at 35° C. andthe samples kept in the autosampler at 4° C. The flow rate was 0.5mL/min, and injection volume 10 μL. The mass spectrometer was an ABQTRAP 5500 (Applied Biosystems SCIEX, Foster City, Calif.) withelectrospray ionization (ESI) source in multiple reaction monitoring(MRM) mode. Sample analysis was performed in positive/negative switchingmode. Declustering potential (DP) and collision energy (CE) wereoptimized for each metabolite by direct infusion of reference standardsusing a syringe pump prior to sample analysis. The MRM MS/MS detectorconditions were set as follows: curtain gas 30 psi; ion spray voltages5000 V (positive) and −1500 V (negative); temperature 650° C.; ionsource gas 1 50 psi; ion source gas 2 50 psi; interface heater on;entrance potential 10 V. In total, 69 water soluble endogenousmetabolites were confidently detected above the baseline set bycell-free samples. Dwell time for each transition was set at 3 msec.Cell samples were analyzed in a randomized order, and MRM data wasacquired using Analyst 1.6.1 software (Applied Biosystems SCIEX, FosterCity, Calif.).

Chromatogram review and peak area integration were performed usingMultiQuant software version 2.1 (Applied Biosystems SCIEX, Foster City,Calif.). Although the numbers of cells were similar and each sample wasprocessed identically and randomly, the peak area for each detectedmetabolite was normalized against the protein content of that sample tocorrect any variations introduced from sample handling throughinstrument analysis. The normalized area values were used as variablesfor the multivariate and univariate statistical data analysis. Thechromatographically co-eluted metabolites with shared MRM transitionswere shown in a grouped format, i.e., leucine/isoleucine. Allmultivariate analyses and modeling on the normalized data were carriedout using SIMCA-P (version 13.0.1, Umetrics, Umei, Sweden). Thepre-processed datasets were evaluated by unsupervised hierarchicalclustering with complete-linkage method.

Example 8: Dual Imaging Methods with PET and Fluorescence Imaging

1. Development of Ultra-pH Sensitive (UPS) Nanoprobes with FluorescentReporter

Recently ICG-functionalized UPS nanoprobes were developed by theinventors with a pH transition at 6.9. PEG-b-PEPA was synthesized usingatom-transfer radical polymerization method with varying repeating unitsin the PEPA segment (40-120, FIG. 70A). ICG, an FDA approved nearinfrared dye was then conjugated to the PEPA segment with different dyedensities (1, 3 and 6 ICGs per polymer chain). At blood pH (7.4) orinterstitial pH (7.2) of normal tissues, the I-UPS_(6.9) nanoproberemains silent (FIG. 70D) as a result of homoFRET-induced fluorescencequenching (Zhou et al., 2012 and Zhou et al., 2011). At these pHs,UPS_(6.9) was present as self-assembled micelles with a diameter of25.3±1.5 nm by dynamic light scattering analysis and a sphericalmorphology by TEM (data not shown). The effect of polymer chain lengthand ICG density on the transition pH, sharpness of response,fluorescence activation ratio and diameter of the nanoprobes wereinvestigated. FIG. 70B shows a representative study on varying PEPAsegment length. Data show increasing the repeating unit of PEPA from 40to 120 resulted in sharper pH transitions (e.g. ΔpH_(ON/OFF) decreasedfrom 0.30 to 0.13, respectively) and slightly lower pH transitions (from6.96 to 6.91, respectively). The particle size also increased with PC7Alength (15 to 30 nm). Three ICGs per polymer chain allowed the mostoptimal dye density with high fluorescence activation ratio and brightfluorescence intensity at the on state. Based on these data, the UPScomposition with 100 repeating unit of PC7A and 3 ICG dyes per chainwere selected. The resulting UPS nanoprobes have sharp pH transition(ΔpH_(ON/OFF)=0.15), high fluorescence activation ratio (>100 foldbetween on and off states) (FIG. 70C), and optimal particle size (25 nm)for tumor penetration. Using this strategy broad tumor specificity withlarge tumor-to-normal tissue ratio in a broad set of animal tumors withdiverse cancer types and organ sites was demonstrated (Wang et al.,2014). Tumor-specific imaging was accomplished in tumors as small as 1mm³. Additionally, the I-UPS_(6.9) nanoprobe is stable in serumcontaining (20% FBS) medium over 48 hours and maintains a sharp pHresponse and high fluorescence activation ratio (FIGS. 70B-70D)

2. Introduce ⁶⁴Cu as the Radioactive Tracer to UPS Nanoprobes for PETImaging

Comparing to other nonstandard PET nuclides, ⁶⁴Cu (t_(1/2)=12.7 h;β+0.653 MeV, 17.4%) has been widely used in many imaging agents based onnanoparticles, antibodies and peptides due to is low positron range,commercial availability, and reasonably long decay half-life (Rossin etal., 2008 and Haubner & Wester, 2004). The stability between the metaland the chelator is important to the outcome of the radiopharmceuticalmodality design. Many chelators have been developed as the chelatingligands for ⁶⁴Cu such as 1,4,7,10-tetraazacyclododecane-tetraacetic acid(DOTA) and 1,4,7-triazacyclononane-triacetic acid (NOTA) and etc (Wadaset al., 2007). Among them, CB-TE2A (FIG. 71) has been reported by Sunand coworkers to form one of the most stable complexes with ⁶⁴Cu (Sun etal., 2002), and the Cu(II)-CB-TE2A complex is more resistant toreductive metal loss than are other tetramacrocyclic complexes (Woodinet al., 2005). CB-TE2A will be used as the chelator for induction of⁶⁴Cu to the UPS. An NHS ester functionalized CB-TE2A will be preparedfollowing the procedure reported in the paper (Liu et al., Angew ChemInt Ed Engl., 48:7346-7349, 2009). On the nanoprobe side, primary aminogroup will be introduced to the PEG terminal of the polymer chains.NH₂—PEG-PC7A will be synthesized by the route described in FIG. 71.Commercially available Fmoc-PEG-OH will be used to make themacroinitiators for ATRP. After deprotection of the polymer after ATRP,the primary amino groups can be regenerated and used for the conjugationto the NHS ester functionalized CB-TE2A. The hybrid micelles will beformed from a mixture of CB-TE2A-PEG-PC7A and PEG-PC7A-ICG by sonicationand solvent evaporation method. The hybrid micelles will be labeled with⁶⁴Cu by incubating ⁶⁴CuCl₂ and micelles in buffered solution followed byultrafiltration.

3. Compare the Imaging Efficacy of Dual Modality UPS and PET with FDGOnly.

Preliminary results in orthotopic HN5 head and neck tumor-bearing miceshowed strong false positive signals from interscapular BAT in two outof three mice, while I-UPS fluorescence delineated tumors with highspecificity (FIG. 72). Clinically, BAT or tensed neck muscles in headand neck cancer patients can lead to misinterpretation as abnormality inPET imaging due to elevated glucose consumption. By introduce ⁶⁴Cu tothe UPS nanoprobes, the distribution of the PET nuclides is anticipatedto be shifted by targeting tumor acidosis and therefore eliminate thepotential false positives from PET with FDG.

In order to compare whether dual modality UPS can provide more accuratetumor detection over FDG by PET scan, activated BAT will be used as amodel to evaluate the imaging efficacy. After an orthotopic head andneck tumor models in mice is established, the tumor bearing mice willcold treated before PET imaging to active BAT (Wang et al., 2012). To bespecific, the tumor bearing mice will be fasted 12 h and placed in apre-chilled cage in a 4° C. cold room for 4 h before PET imaging. Themice will be evenly divided into three groups and will be injected withthe following agents respectively through tail vein: 1) FDG; 2) dualmodality UPS; 3) propranolol and FDG. Propranolol is a β adrenoceptorinhibitor which will suppress BAT activation and serve as the negativecontrol. PET images will be acquired and reconstructed into a singleframe using the 3D Ordered Subsets Expectation Maximization (OSEM3D/MAP)algorithm. Regions of interest (ROI) will be drawn manually encompassingthe tumor/BAT in all planes containing the tissue. The target activitywill be calculated as percentage injected dose per gram (% ID/g).Standardized uptake value (SUV) will also be calculated for tumors,interscapular BAT as well as surrounding normal tissues for evaluationof potential false positives. Histology will serve as the gold standardfor verdict of the presence of cancerous tissue or BAT. All tissueswhich show a positive signal in head and neck region from either FDGgroup or UPS group will all be collected for paraffin embedding andsectioning. H&E staining will be prepared from these slides forhistology validation to correlate with the results from each group. Eachspecimen will be assigned as FDG+/− (from PET), ⁶⁴Cu-UPS+/− (from PET),cancer cell+/− (from histology) and BAT+/− (from histology). Statisticalanalysis will be used to judge whether dual modality UPS significantlyimprove detection accuracy.

Example 9: Cancer Surgery and Tumor Removal Process with UPS Nanoprobes

1. Broad Cancer-Specific Imaging of Multiple Tumor Types with UPSNanoprobes

One advantage of the I-UPS design is its compatibility with existingoperating room camera systems that have already been approved forICG-based imaging in open surgery (SPY Elite® by Novadaq), microsurgery(Leica, Carl Zeiss), laparoscopy (Karl Storz, Olympus), and roboticsurgery (da Vinci®), lowering barriers for clinical translation. Usingthe SPY camera, the feasibility of the I-UPS_(6.9) nanoprobe to imagetumor acidosis in multiple cancer types was investigated, including thehead and neck (human HN5, FaDu and HCC4034 orthotopic xenografts in SCIDmice; HCC4034), breast (human MDA-MB-231 in SCID mice and murine 4T1 inBALB/C mice), kidney (human orthotopic XP296 tumors in SCID mice), brain(human glioblastoma U87 xenograft), and peritoneal metastasis from theGI tract (human colorectal HCT-116 tumors in SCID mice, FIG. 73).Results show high tumor/normal tissue contrast (T/N ratio >20) in thisbroad set of tumors. In particular, I-UPS signals were lacking intypical false positive tissues in the head and neck (e.g., brown fat) aswell as brain parenchyma (likely due to the blood-brain-barriers(Hawkins & Davis, 2005 and Kreuter, 2001) that prevent UPS uptake).These results demonstrate the robustness of extracellular acidic pH as acancer target and the broadly applicable and cancer-specific detectionby the I-UPS nanoprobes.

Example 10: Dual Fluorescence Reporter UPS Nanoprobes

1. UPS Nanoprobes with Dual Fluorescence Reporters

To independently evaluate nanoprobe dose and pH activation in tumoracidosis imaging, UPS nanoprobes with a dual fluorescence reporter willbe constructed: an “Always ON” reporter to track nanoparticledistribution regardless of pH, and a pH-activatable reporter. Initialattempts at conjugating a dye (e.g., Cy5.5) to the terminal end of PEO(such as the surface of UPS nanoprobes) succeeded in an Always ONsignal, however, the resulting nanoparticles were unstable because ofdye binding to serum proteins. To overcome this limitation, a heteroFRETdesign using a pair of fluorophores that are introduced in the core ofthe micelles will be employed. For example, a FRET pair (e.g., BODIPYand Cy3.5 as donor and acceptor, respectively) were separatelyconjugated to the PR segment of the UPS_(6.9) copolymer. Mixing of thetwo dye-conjugated copolymers (optimal molar ratio ofdonor/acceptor=2:1) within the same micelle core allowed theheteroFRET-induced fluorescence quenching of the donor dye (e.g.,BODIPY, λ_(ex)/λ_(em)=493/503 nm) in the micelle state (pH>pK_(a)), butfluorescence recovery in the unimer state after micelle disassembly atlower pH (FIGS. 76A-C). To generate the “always ON” signal, the weightfraction of Cy3.5-conjugated copolymer in the micelles was kept low(e.g., 40%) to avoid homoFRET-induced fluorescence quenching for theacceptor dye (Cy3.5, λ_(ex)/λ_(em)=591/604 nm) in the micelle state(Zhou et al., 2011; 2012). The resulting UPS nanoparticle show constantfluorescence intensity in the Cy3.5 channel across a broad pH range,while achieving ultra-pH sensitive activation at 6.9 of the BODIPYsignal (FIG. 75). Since both fluorophores are embedded within themicelle core, the resulting UPS nanoparticles are stable and free fromprotein fouling.

In the current study, the heteroFRET design and BODIPY/Cy3.5 pair willbe employed to introduce Always-ON/OFF-ON dual reporters in the UPSnanoprobes. After micelle formation, the nanoprobes will first becharacterized by dynamic light scattering (DLS, Malvern ZetasizerNano-ZS model) for hydrodynamic diameter (D_(h)) and zeta-potential.Size and morphology of UPS nanoprobes will be further analyzed bytransmission electron microscopy (TEM, JEOL 1200 EX model) andcorrelated with DLS results. For study of fluorescence activation inresponse to pH, micelles will be prepared in different pH buffers (pHwill be controlled from 6.0 to 7.4 with 0.1 pH increment) at aconcentration of 0.1 mg/mL. The nanoprobes will be excited atcorresponding wavelengths of the fluorophores on a Hitachi fluorometer(F-7500 model), and the emission spectra will be collected. The emissionintensity will be used to quantify the ON/OFF ratio. The criticalmicelle concentration (CMC) will be measured using the pyrene method(Kalyanasundaram & Thomas, 1977 and Winnik, 1993). Stability of the dualreporter UPS nanoprobes in fresh mouse serum will also be tested aspreviously described (Wang et al., 2014).

2. UPS Nanoprobes with pH Transitions from 6.5 to 7.1.

A finely tunable series of UPS nanoprobes from 6.5 to 7.1 will besynthesized to target tumor pH_(e) with different degrees of acidosis. Arandom copolymer strategy for the construction of a UPS library withoperator-predetermined pH transitions (4.0-7.4) and sharp pH response isreported herein and in (Ma et al., 2014). Three design criteria must bemet: (1) In the PEO-b-PR copolymer, a random PR block (P(R₁-r-R₂), whereR₁ and R₂ are monomers with different alkyl chain lengths on thetertiary amine) must be used to ensure a single pH transition. A blockedPR segment (P(R₁-b-R₂)) resulted in two pH transitions reflecting thedifferent ionization behaviors of the R₁ and R₂ blocks; (2) monomerswith closely matched hydrophobicity in R₁ and R₂ are necessary toachieve sharp pH response. In one non-limiting example, the ΔpH_(OFF/ON)is <0.25 pH when adjacent akyl groups are used (e.g.,R₁/R₂=ethyl/propyl) whereas ΔpH_(OFF/ON) is >0.5 pH when R₁/R₂ areethyl/pentyl groups; (3) the hydrophobicity of P(R₁-r-R₂) segment can befine-tuned by controlling the molar fraction of R₁ and R₂ monomers,which leads to precisely controlled transition pH.

Based on the above criteria, a series of PEO-b-P(DEA_(x)-r-DPA_(y))copolymers with varying ratios of the two monomers, diethylaminoethylmetharylate (DEA-MA) and diisopropylaminoethyl metharylate (DPA-MA)(FIG. 76A) will be synthesized. The total repeating unit will becontrolled at 100 (x+y=100). Aminoethyl metharylate (AMA-MA) will beintroduced (z=3 per chain) for fluorophore conjugation. FIG. 76B showsthe data on pH-dependent UPS activation using Cy5.5, a representativedye. Results indicate a finely tuned series of UPS nanoprobes in the pHrange of 6.3 and 7.8. All nanoprobes maintained the sharp pH response(ΔpH_(OFF/ON)<0.25). Plot of transition pH vs. DPA molar percentage(quantified by ¹H NMR) shows a linear correlation (FIG. 76C). Thiscorrelation will be used as a standard curve to determine thecomposition of PEO-b-P(DEA_(x)-r-DPA_(y)) copolymers to target pHtransitions at 6.5, 6.7, 6.9 and 7.1. For each transition pH, ananoprobe with ICG conjugation for SPY imaging or dual fluorescencereporters for mechanistic studies will be produced.

Example 11: Analysis of pH Regulatory Mechanisms in Tumor Progression

1. pH Regulatory Mechanism of Tumor Acidosis in Different GlycolyticPhenotypes at Different Stages of Tumor Progression

Using the dual reporter UPS nanoprobes described herein, tumors withdivergent glycolysis propensity will be investigate to determinedifferent pH regulatory pathways employed to achieve tumor acidosis.More specifically, whether highly glycolytic tumors will predominantlyemploy monocarboxylate transporters (e.g., MCT1/4) for pH regulationwhereas glycolysis-impaired tumors utilize carbonic anhydrase IX intumor acidosis will be investigated. Competent glycolysis head and neckcancer cells (e.g., HN5 or FaDu) will be used as positive controls, andcreate isogenic, glycolysis-impaired tumors by stable knockdowns of keyglycolytic enzymes (e.g., LDHA or PKM2). Previous studies have shownthat shRNA knockdown of LDHA or PKM2 selectively inhibits glycolysis andreprograms the cells toward the OXPHOS pathway (Christofk et al., 2008;Fantin et al., 2006). Small molecular inhibitors (e.g., suicideinhibitor for MCT1/4, or aryl sulfonamides for CAIX) will be used incombination with immunohistochemistry of MCT1/4 and CAIX. The pattern ofUPS activation will be correlated with the spatial expression of pHregulatory proteins in tumor sections.

2. Mechanistic Investigation of Tumor Acidosis by Perturbation withSmall Molecular Inhibitors

Tumor bioenergetics involves enhancement of glycolytic machinery ormitochondrial oxidative phosphorylation (OXPHOS) pathways. Severalmolecular mechanisms are responsible for maintaining an alkaline pH_(i)in cancer cells and acidic pH_(e) in tumor microenvironment (FIG. 77).The pH regulatory machinery involves the interplay of multiple proteins,including monocarboxylate transporters (MCT1 and MCT4) (Enerson &Drewes, 2003 and Halestrap & Price, 1999), carbonic anhydrases (CAIX andCAXII) (Supuran, 2008 and Supuran, 2010), anion exchangers (AE1, 2 and3) (Sterling, et al., 2002; Morgan, 2004), Na⁺-bicarbonate exchangers(NBCs) (Pouyssegur et al., 2006), Na⁺/H⁺ exchangers (NHEs) (Pouysseguret al., 2006), and V-ATPase (Perez-Sayans et al., 2009).

To examine the acidosis mechanism in tumors with different glycolyticphenotypes, the inventors will first start with inhibitors of the twomain regulatory proteins in the acidosis process: suicide CHC inhibitorfor MCT1/4 (FIGS. 78A-78E) and acetazolamide for CAIX. The dual reporterUPS nanoprobes described herein will be administered intravenously toperturb the pH regulation in the tumor microenvironment and use the dualreporter UPS nanoprobes for imaging spatiotemporal response ofacidification. Three groups of mice will be used with subcutaneous lungtumors that have different glycolysis phenotypes: 1) orthotopic HN5 orFaDu tumors with competent glycolysis rates; 2) orthotopic HN5 or FaDutumors with impaired glycolysis by shRNA knockdown of LDHA (Fantin etal., 2006). The tumor size will be controlled at ˜200 mm³ for allgroups. The mice from each group will then be divided into foursub-groups. Each sub-group of mice will receive: 1) PBS; 2) CHCinhibitor; 3) acetazolamide or 4) both CHC inhibitor and acetazolamide,respectively followed by administration of dual reporter UPS. At 1, 2,4, 12 and 24 h post UPS injection, the BODIPY (OFF-ON) fluorescenceintensity (FI) and Cy3.5 distribution (Always ON) from each tumor willbe measured by a Maestro small animal imaging system (PerkinElmer) andquantified by ImageJ. By comparing the FI(subgroup1) among the threedifferent groups, whether cancer cells with divergent glycolysis ratesall produce acidic pH_(e) in the tumor microenvironment will bedetermined. By calculating the ratio of FI(subgroup2/3/4)/FI(subgroup1)from the same group, how each pathway contributes to the overallacidosis for the tumors with different glycolysis rates will bedetermined. At the end of each experiment, tumors will be collected andfrozen sections will be prepared from the specimen. For each tumorsection, a BODIPY image for activated nanoprobes and a Cy3.5 image forabsolute probe distribution will be captured. The relative contributionof MCT1/4 or CAIX to the tumor acidosis will be normalized from theratio of activated ON/OFF BODIPY vs always ON Cy3.5 fluorescentintensity. The frozen section will be stained by H&E or antibodies tocorrelate with the expression profiles of MCT1/4 or CAIX in tumors.Hypoxic biomarker HIF1α will be also stained to compare the distributionpatterns with activated nanoprobes.

3. Investigate the Intratumoral Heterogeneity of Tumor Acidosis atDifferent Stages of Tumor Progression.

It is known that a continuum of bioenergetic remodeling exists alongtumor progression (Jose et al., 2011). Small tumors have a tendency oflow conversion of glucose to lactate but relatively high conversion ofglutamine to lactate, whereas large tumors have high glucose and oxygenutilization rate despite low oxygen and glucose supply (Eigenbrodt etal., 1998). Data show that the I-UPS method can detect very small tumorfoci (<1 mm or one million 4T1 cancer cells in BalB/C mice, FIG.79A-79C) as a bright punctate under SPY camera. This result demonstratesthat I-UPS has the adequate sensitivity to detect small tumor nodules atan early stage of development.

To monitor the potential switching of tumor acidosis mechanism duringtumor growth, HN5 and HN5 glycolysis-impaired models will be studied andevaluate the nanoprobe activation at different stages of tumor growth.When the tumors grow to sizes of 10, 100, 500 and 1000 mm³, the animalswill first be imaged without injection of MCT1/4 or CAIX inhibitors.Afterwards, CHC inhibitor or acetazolamide will be injectedintravenously to block the corresponding pH regulation pathway and theanimals will be imaged again to compare the fluorescence intensitybefore and after perturbation. The percentage decrease in fluorescenceintensity as a result of CHC inhibitor or acetazolamide will bequantified and correlated with the expression levels of MCT1/4 or CAIXin tumor sections, respectively. Vasculature (anti-CD31) and hypoxia(pimonidazole) stains will also be performed to assess impact of thevascularization and hypoxia on UPS activation at different stages oftumor progression as described in (Wang et al., 2014).

Example 11: UPS Imaging in Detecting Tumors with Divergent GlycolyticPhenotypes

1. Mouse Tumor Models with Divergent Glycolysis Rates.

In one series, orthotopic head and neck tumors (HN5, FaDu, or HCC4034,FIG. 73) will be established and their isogenic, glycolysis-impairedtumors by stable knockdowns of LDHA described herein. In another series,several non-small cell lung cancer cell lines with constitutively highvs. low glycolysis rates from a panel of 80 cell lines previouslyestablished will be selected. Using these animal models with divergentglycolytic phenotypes, the hypothesis that acidosis imaging by I-UPSnanoprobes allows higher cancer specificity will be tested particularlyfor glycolysis-impaired tumors over FDG-PET. In addition, the falsepositive signals from both imaging methods in normal tissues will beinvestigated.

2. Comparison of I-UPS and FDG-PET Imaging in Normal andGlycolysis-Impaired Tumor Models of the Head and Neck.

A series of orthotropic head and neck tumor models in mice withcompetent glycolysis and impaired glycolysis will be established.Specifically, 10⁶ of selected head and neck cancer cells (HN5, FaDu orHCC4034) will be injected in the submental triangle region in SCID miceand let tumors grow to ˜200 mm³. The mice will be divided into twogroups: one group will be injected with scrambled short hairpin RNA(shRNA_(scr)) as the competent glycolysis group; the other group will beinjected with lactate dehydrogenase (LDHA) knock-down short hairpin RNA(shRNA_(LDHA)) (Fantin et al., 2006) to block lactate formation as theglycolysis-impaired group. Mice will be fasted for 12 h prior to PETimaging. Each mouse will receive 150 μCi of FDG in 150 μL in salineintravenously via tail vein injection. PET images will be acquired onehour post-injection for 15 mins. PET images will be reconstructed into asingle frame using the 3D Ordered Subsets Expectation Maximization(OSEM3D/MAP) algorithm. Regions of interest (ROI) will be drawn manuallyencompassing the tumor in all planes containing the tissue. The targetactivity will be calculated as percentage injected dose per gram (%ID/g). Standardized uptake value (SUV) will also be calculated fortumors as well as surrounding normal tissues and other organs ofinterests (e.g., brain, kidney, heart, and tonsil) for evaluation ofpotential false positives. Preliminary data in orthotopic HN5 head andneck tumor-bearing mice showed strong false positive signals from brownadipose tissues (Christofk et al., 2008; Fantin et al., 2006) in two outof three mice, while UPS detected tumors with high specificity (FIG.80). Clinically, interscapular brown adipose tissue or tensed neckmuscles in head and neck cancer patients can also lead tomisinterpretation as abnormality in PET imaging due to elevated glucoseconsumption (Mirbolooki, 2011; Wang et al., 2012). Histology in thenormal tissues will be preformed to verify the tissue origin of thefalse positive signals.

After PET imaging, mice will be kept overnight to deactivate theradioactive tracer (¹⁸F, t_(l/2)=110 min). I-UPS (2.5 mg/kg) will thenbe injected through the tail vein. Mice will be imaged using a SPYElite® surgical camera 24 hours after injection. After whole bodyimaging, mice will be dissected to remove major organs (e.g., heart,liver, kidney, lung, brain, spleen, etc.). Ex vivo imaging of tumors andnormal tissues will be imaged by the SPY Elite® surgical camera. Tumorto normal tissue ratio (T/N) will be quantified using Image J softwarefor all fluorescent images. Between the two divergent glycolytic animalgroups, how the glycolysis degrees impact % ID/g or SUV for PET imagingand T/N for fluorescent imaging will be compared. After fluorescenceimaging, the major organs of the animals will be frozen sectioned andstained with H&E. A clinical pathologist will verify the presence ofmalignance and tissue origin of false positive signals.

3. Investigation of I-UPS Imaging Specificity in Non-Small Cell LungTumor Models.

In addition to head and neck cancer models, the imaging specificity ofI-UPS and FDG-PET in selected lung cancer models with divergentglycolysis rates will also be investigated. As part of the lung cancerSPORE, the glucose consumption rate and lactate secretion rate for apanel of over 80 non-small cell lung cancer (NSCLC) cells in cellculture (FIG. 81) has been previously quantified. Plot of glycolysisrates (Lactate_(out)/Glucose_(in)) vs cell lines illustrates cellautonomous, constitutively divergent glycolytic phenotypes across theNSCLC cells. Based on this data, two groups of lung cancer cells withhigh and low glycolysis rates will be selected. Specifically, H2170,HCC515 and H2347 will be chosen as the high glycolytic panel(Lactate_(out)/Glucose_(in)˜2.0), and H228, H1755 and HCC78 will bechosen as the low panel (Lactate_(out)/Glucose_(in)<0.5). These celllines are available from the UTSW/MDACC lung SPORE and have been shownto produce subcutaneous tumors in SCID mice. In a typical procedure,2×10⁶ lung cancer cells will first be injected subcutaneously at theleft flank of the mice to form tumor xenografts in SCID mice. When thetumors grow to −200 mm³, the mice will be imaged with PET first,followed by fluorescent imaging after injection of I-UPS nanoprobesusing the procedures described above. The I-UPS imaging outcomes will becompared with those from FDG-PET. In particular, whether I-UPS imagingwill stay silent in verified false positive tissues from FDG-PET (e.g.,brown fat, thoracic muscle by histology) will be evaluated and alsowhether I-UPS imaging is able to illuminate lung tumors withconstitutively low glycolysis rates that are potentially undetected byFDG-PET.

Example 12: Optimize UPS Nanoprobe Activation Relative to MicroscopicTumor Margins in Cancer Tumors

1. Quantify UPS Activation Profile and Correlate with Microscopic TumorMargins.

For selected tumor models, nanoprobes with the Always ON/OFF-ON dualreporter will be injected intravenously. Starting at 15 mins, 1, 4 and24 h post-injection, tumor and surrounding tissue will be collected andfrozen sections will be prepared from the specimen.

For each tumor section, a BODIPY image will be captured for activatednanoprobes and a Cy3.5 image for absolute probe distribution. The frozensection will be stained by H&E to identify the true tumor margin(current clinical gold standard). For larger tumors, multiple imageswill be captured and stitch them together for holistic comparison.

For quantitative image analysis, (1) a tangent line from a true tumormargin point (zero point) identified by the histology image will bedrawn (FIG. 82), (2) draw another line perpendicular to the tangentline, (3) quantify BODIPY and Cy3.5 fluorescence intensity (e.g., ±500μm from the zero point) along the perpendicular line using ImageJprogram, (4) repeat steps 1 to 3 from multiple margin points for eachtumor slice and average multiple linear profiles, and (5) plot averagedfluorescence intensity vs. distance in the BODIPY and Cy3.5 channels.FIG. 82 illustrates the schematic of margin analysis by the dualreporter nanoprobes.

By comparing the H&E image and BODIPY (pH activatable reporter) map, theprobes will be determined to see if the probes can delineate the tumormargin through pH activation by glycolytic cancer cells. Some specificquestions that will be addressed include: (1) what is the distributionand nature of the lactate secreting cells and is lactate secretion andthus the accuracy of the margins affected by tumor size, type, and stageand (2) whether UPS nanoprobes will be able to discriminate pHheterogeneity within and/or across tumor borders and whether residualcancer cells infiltrating into the normal tissue can be detected beyondthe margin due to the lack of EPR effect. By comparing the CFP and Cy3.5(always ON reporter) maps, the distribution of the probes inside thetumor will be determined vs. the normal tissue around the tumor overtime. By comparing BODIPY and Cy3.5 signals, the probe activation(I_(BODIPY)/I_(Cy3.5)) will be determined relative to probe accumulation(I_(Cy3.5)). Without wishing to be bound by any theory, it is believedto determine whether dose accumulation via EPR effect or pH activationdrives margin delineation. This set of curves for a series of dualreporter probes with different pH transitions from 6.3 to 7.1 willestablish and investigate whether tuning the pH transition changes thesensitivity and specificity of tumor margin delineation. The optimalI-UPS is universal will be examined or is dependent on the type and/orsize of the tumor. Finally, CFP-labeled cancer cells will be used tofurther test the sensitivity and specificity of the probes for thedetection of cancer cells infiltrating into the normal tissue beyond themargin.

2. Antitumor Efficacy and Long-Term Survival Studies.

Orthotopic tumor xenografts (HN5 and FaDu for head and neck cancer, 4T1and MD-MBA-231 for breast cancer) will be used to evaluate the antitumorefficacy of I-UPS-guided resections. For each study, I-UPS nanoprobeswill be intravenously injected 24 h before surgery. The animals will bedivided into 4 groups (n=10 or 15 for each group): (1) no surgery; (2)tumor debulking control (where visible tumor is partially removed); (3)white light surgery with complete removal (based on surgeon's bestestimation) and (4) SPY-guided tumor resection. These experimentalgroups will allow exploration of the difference between conventionalsurgery under white light and fluorescent surgery. Pilot studies havebeen performed using the I-UPS_(6.9) probes (FIGS. 74A & 74B and78A-78E). Similar experiments for optimized I-UPS probes in additionaltumor models (e.g., FaDu and MDA-MB-231) will be carried out.

After surgery, the Kaplan-Meier survival curves will be determined tocompare the antitumor efficacy between each group. For all resectedanimals, tumor occurrence at the primary site will be examined andrecorded. In addition, the effects of surgery on the swallowing functionof the mice will be estimated. Without wishing to be bound by anytheory, it is believed that the greater the amount of normal tissue thatis removed during tumor extirpation, the greater the resultantfunctional deficit to the animal and therefore swallowing. The mice willbe weighed both pre- and post-operatively. Daily weights will berecorded post-operatively for 1 week and twice a week thereafter.Percentage body weight lost will be used as a proxy for feeding andswallowing function. Weights will be normalized to the initial weight toaccount for animal growth.

Example 13: Biological Profile and Pharmcokinetics

1. Pharmacokinetic/Biodistribution (PK/BD) Studies.

Previous studies using ³H-labelled PEG-b-PC7A (UPS_(6.9)) and PEG-b-PDPA(UPS_(6.3)) show that the resulting UPS nanoparticles have significantlydifferent PK/BD profiles (FIG. 83) despite similar hydrodynamicdiameters (25.3±1.5 vs. 24.9±0.8 nm, respectively), zeta potential(−0.7±1.1 vs. −3.5±0.6 mV), and PEG length (both 5 kD). The α-phasehalf-lives were 1.0±0.2 and 4.3±0.7 h (P<0.05), and β-phase half-liveswere 7.5±0.3 and 19.6±2.1 h (P<0.01) for UPS_(6.9) and UPS_(6.3)nanoparticles, respectively. Biodistribution studies at 24 h afternanoparticle injection showed liver and spleen were the major organs forthe clearance of both nanoparticles. The faster clearance of UPS_(6.9)over UPS_(6.3) was attributed to its higher transition pH and highersusceptibility in UPS activation and clearance from blood.

In this study, the PK/BD studies will first be perform for the optimizedI-UPS composition described herein using ³H-labelled copolymers aspreviously established (n=5 for each group). Blood will be collected at2 min, 0.5, 1, 3, 6, 12 and 24 h after injection. At the end of theexperiment, animals will be sacrificed and tumor tissue and major organs(heart, liver, spleen, kidney, etc.) will be removed. Dissected organswill be weighed, homogenized and treated with scintillation mixtures.Both the blood and tissue samples will be quantified by a liquidscintillation counter (Beckman LS 6000 IC). The UPS distribution indifferent organs/tissues will be calculated as the percentage ofinjected dose per gram of tissue. In addition to blood and tissuesamples, urine and feces samples will also be collected to analyze theclearance of I-UPS via kidney secretion and GI tract. These experimentswill be performed in metabolic cages in a designated animal facility oncampus.

2. Assessment of Innate Immunity Response.

To evaluate whether I-UPS may cause strong innate immunity, I-UPSnanoprobes will be intravenously injected at 1×, 10× and 50× of theimaging dose in immunocompetent C57BL/6 mice (n=5 for each group). At 2,6 and 24 h, blood samples (100 μL) from the tail vein will be collected.Serum will be separated and the cytokine profiles analyzed. The currentLuminex™ multiplex assay can detect 23 cytokines (e.g., IFN-α and -β,IL-2, IL-4, IL-12, IL-17, etc) from 25 μL of serum. PBS will be used asa negative control. If a significant increase is observed in cytokines,more detailed analysis on immune response will be performed (e.g.,examining neutrophil or other leukocyte production, complementactivation, inflammatory response in the spleen and lymph nodes) overlonger time frame such as 2-4 weeks.

Example 14: UPS Nanoparticles Containing Multiple Different Polymers

1. Use of Micelles with Multiple Different Polymers

Initially, a series of amphiphilic block copolymers PEG-b-PR, where PEGis poly (ethylene glycol) and PR is an ionizable segment (Scheme 1 andTable 12) were synthesized

TABLE 12 Characterization of PEG-b-(PR-r-AMA₃) diblock copolymers.M_(n), ¹H-NMR M_(n), GPC M_(w), GPC Copolymer (kD)^(a) (kD)^(b) (kD)^(b)PDI^(b) PEG₁₁₄-b-P(EPA₇₅-r- 20.8 20.1 24.9 1.24 AMA₃)PEG₁₁₄-b-P(DPA₈₁-r- 22.8 22.3 25.6 1.15 AMA₃) PEG₁₁₄-b-P(DBA₇₅-r- 23.522.8 26.5 1.16 AMA₃) ^(a)Number-averaged molecular weight (M_(n)) asdetermined by ¹H-NMR. ^(b)Number-averaged (M_(n)), weight-averagedmolecular weight (M_(w)), and polydispersity index(PDI = M_(w)/M_(n))were determined by GPC using THF as the eluent.

The PEG-b-PR copolymers were encoded with different fluorophores. Threeexemplary PEPA-BDY493, PDPA-TMR, and PDBA-Cy5 fluorescent polymers wereselected and characterized in terms of dye conjugation number andefficiency as well as quantum yield (FIG. 84B and Table 13). ThePEPA-BDY493, PDPA-TMR, and PDBA-Cy5 nanoprobes had pH transitions at6.9, 6.2, and 5.3, which cover the pH changes during endocytic pathwayfrom clathrin-coated vesicle (CCV) to early endosome (pH˜6.0), then tolate endosome/lysosome (pH˜5.0-5.5) (Huotari & Helenius, 2011). Theparticle sizes of these nanoparticles were 25-35 nm with narrowdistribution. The fluorecent activation ratios (RF) were 30, 91, and 107fold for PEPA-BDY493, PDPA-TMR, and PDBA-Cy5 nanoprobes with sharp pHresponse (ΔpH₁₀₋₉₀%=0.18-0.22, Table 14 and FIGS. 85-88). Using thesonication method, a hybrid UPS nanoprobe system consists of threecomponents PEPA-BDY493, PDPA-TMR, and PDBA-Cy5 was engineered, as shownin FIGS. 84A-84C. Without wishing to be bound by any theory it isbelieved that the three fluorescent polymers will self-assembled into ahomogenous hybrid UPS nanoprobe at higher pH. After endocytosis, thehybrid UPS nanoprobe will sequentially disassemble and fluoresce at theindividual pH_(t) of each polymer (e.g., 6.9, 6.2, 5.3) to tract theendosomal maturation process associated with subtle pH changes at singleorganelle resolution in living cells (FIG. 84C).

TABLE 13 Measurement of conjugation efficiency and quantum yields ofdye-conjugated copolymers. Dye conjugation Quantum yield (Φ_(F))^(a)Efficiency Free Conjugated PR-Dye Number (%) dye^(b) dye Mixture^(d)PEPA-BDY493 2.2 73 0.90 0.05/0.68^(c) 0.87 PDPA-TMR 2.1 70 0.68 0.260.65 PDBA-Cy5 2.2 73 0.28 0.24 0.27 ^(a)In methanol unless notedotherwise. ^(b)Obtained from literature. ^(c)In methanol with 0.5% 1MHCl. ^(d)Mixture of free dye with dye-free PDPA copolymer.

TABLE 14 Characterization of PEG-b-(PR-r-Dye) nanoprobes. Particle sizeR_(F) Copolymer (nm) pH_(t) ΔpH_(10-90%) (F_(on)/F_(off)) ^(a)PEPA-BDY493 24.65 ± 1.55 6.95 0.22 30 PDPA-TMR 30.15 ± 2.15 6.20 0.18 91PDBA-Cy5 35.49 ± 2.92 5.26 0.20 107 ^(a) Determined by fluorescenceemission intensity of different dyes.

To demonstrate the formation of homogenous hybrid nanoparticle, a seriesof homoFRET and heteroFRET experiments were designed and performed. ThehomoFRET experiment involves a molecular mixture of one fluorescentPEG-b-PR polymer and another label-free PEG-b-PR polymer with differentpH transitions. In this example, PEPA-Cy5 were used and mixed up withPEPA, PDPA, or PDBA polymer at the molar ratio of 1:19 for the labeledversus label-free polymer. Results showed the successful formation ofmolecularly mixed micelle of PEPA-Cy5 with PEPA, PDPA or PDBA polymer inthe same micelle which indicated by the recovery of the Cy5 fluorescentsignal through overcoming homoFRET effect (FIG. 114A & 114B) (Zhou etal., 2012). In contrast, the micelle mixture of PEPA-Cy5 and anotherlabel-free micelle showed no Cy5 signal recovery. The same result wasalso observed in the moluecularly mixed micelle of PDBA-Cy5 and anotherlabel-free PEG-b-PR polymer (FIGS. 89A & 89B). All these resultsindicated that the PEPA, PDPA, and PDBA polymers can form a homogenoushybrid nanoprobe.

To further verify the formation of the hybrid nanoprobe, thefluorescence transfer effect was examined from copolymers encoded withdifferent hetero-FRET dyes: PEPA-BDY493, PDPA-TMR, and PDBA-Cy5. Tominimize the homoFRET effect, each copolymer was encoded with one dye inthe hydrophobic PR segment. Two or three copolymers were dissolved inTHF and then were added dropwise into water to produce a molecularlymixed micelle as described herein. In the pair of PEPA-BDY and PDPA-TMR(molar ratio=1:1), the fluorescence intensity at BDY493 emissionwavelength (510 nm) in the molecularly mixed micelle decreased over4-fold as compared to PEPA-BDY493 alone micelle solution. Moreover, thefluorescence intensity at TMR emission (580 nm) increased over 4-foldfor mixed micelle solution over PDPA-TMR micelle solution (FIG. 90A).The other three sets of hetero-FRET polymers: (i) PDPA-TMR and PDBA-Cy5,(ii) PEPA-BDY493 and PDBA-Cy5; (iii) PEPA-BDY493, PDPA-TMR and PDBA-Cy5were also extensively investigated (FIGS. 90B-90D). In the set ofPEPA-BDY493, PDPA-TMR and PDBA-Cy5 fluorescent polymers, the sequentialFRET effect from BDY493 to TMR was observed, finally to Cy5 dye (FIG.90D). The fluorescence intensity at BDY493 emission in hybrid nanoprobedecreased over 4-fold as compared to PEPA-BDY493 alone micelle solution,while the Cy5 signal increased over 25-fold for hybrid nanoprobe overPDBA-Cy5 micelle. These results clearly demonstrated that the threePEG-b-PR copolymers can self-assembled into a homogenous hybrid UPSnanoprobe.

After demonstrating the formation of hybrid UPS nanoprobe, a hybridnanoprobe system was produced using PEPA-BDY493, PDPA-TMR and PDBA-Cy5fluorescent polymers each PR chain conjugated with ˜2.2 dyes (FIG. 91).The fluorescence emission spectra at different excitation wavelength(485, 545, 640 nm) and different pH (7.4, 6.7, 5.8, and 5.0) werecollected and plotted (FIGS. 92A-92D and FIG. 93A-93F and FIG. 94).Results showed that all the three fluorescent polymer components kept“silent” at the neutral pH. When the pH decreased to 6.7, the PEPA-BDYcomponent was firstly released and activated to produce the greensignal, while the other two components still stayed “OFF”. When the pHwas lowered to 5.8, the PDPA-TMR signal was activated to produce the redsignal and the PDBA-Cy5 component was completely “silent” in this stage.Finally, the PDBA-Cy5 was activated when the solution pH was decreasedto 5.0. In this stage, all three fluorescent polymers were fullyactivated. The particle sizes of hybrid UPS nanoprobe were ˜30-40 nm atpH between 7.4 and 5.8, and dropped to 8.7 nm as unimers at pH 5.0determined by dynamic light scattering analysis (DLS). Similarobservation was made by TEM analysis (FIGS. 95A & 95B). The pH_(t)values for PEPA-BDY493, PDPA-TMR, and PDBA-Cy5 components in hybrid UPSnanoprobe were 6.9, 6.2, and 5.3, which were consistent with theircorresponding single component nanoprobes. Overall, the fluorescenceactivation ratios for PEPA-BDY493, PDPA-TMR, and PDBA-Cy5 were 74, 123,and 30 with sharp pH response (ΔpH₁₀₋₉₀%=0.20-0.25, Supplementary TableS4). Using DLS analysis, the count rates of the hybrid nanoprobe versuspH values were plotted and also observed the multi-stage activationpattern as shown in FIG. 92E. In each stage, one fluorescent polymerwill be released, fluoresce, and finally all the polymers willdissociate indicated by the count rates reached to zero. The multi-stageactivation of hybrid UPS nanoprobe at different pH was also imaged andverified by Maestro CRI imaging system as shown in FIG. 92F.

To check the synchronized cell uptake of three components in hybrid UPSnanoprobe, the UPS nanoprobe was functionalized with 5% Erbitux(humanized EGFR antibody) (Adams & Weiner, 2005) through thiol-maleimidelinkage as described herein. The Erb-encoded hybrid nanoprobe had threedistinct pH transitions at 6.9, 6.2, and 5.3 with ΔpH₁₀₋₉₀% values of0.20-0.25. The fluorescence ON/OFF activation ratios of the hybridnanoprobe were 200, 191, and 35-fold for BDY493, TMR, and Cy5 channels,respectively. To investigate the specificity of Erb-encoded UPSnanoprobe, the A549 cells were incubated with Erb-encoded PDPA-TMRnanoprobe. Fifteen minutes after Erb-encoded PDPA-TMR incubation,punctate fluorescence activation was observed inside the cells. At 1 h,an over 250-fold fluorescence increase in the Erb-encoded PDPA-TMRnanoprobe was observed over PDPA-TMR nanoprobe control group,demonstrating the high specificity to EGFR biomarker (FIG. 96). Afterverifying the specificity of the Erb-conjugated UPS nanoprobe, thesynchronized uptake of Erb-encoded hybrid UPS nanoprobe was checked(FIG. 97). A549 cells were incubated with Erb-encoded hybrid UPSnanoprobe for 3 h, and imaged by a confocal microscope. In the controlgroup, A549 cells were incubated with the cocktail of PEPA-BDY493,Erb-encoded PDPA-TMR, and PDBA-Cy5 three nanoprobes. The synchronizeduptake of Erb-encoded hybrid nanoprobes in single endocytic organellewas observed, while only Erb-encoded PDPA-TMR nanoprobe in the controlgroup was internalized and activated inside the cells. Importantly, allthe punctate blue and red fluorescent dots were colocalized with asubset of green fluorescent dots, indicating that the hybrid nanoprobescan be utilized for the evaluation of endocytic organelle maturation.

To track endosome maturation in real-time, the A549 cells were incubatedwith Erb-encoded hybrid UPS nanoprobe for 30 min at 4° C. to allow forspecific cell binding, then the medium was removed and washed thrice.The intracellular uptake and activation of hybrid nanoprobe at 37° C.was imaged by confocal microscope. As expected, the PEPA-BDY493component was firstly released and activated to produce the greenfluorescent dots at 10 min, and the intensity increased and reached aplateau after 30 min incubation (FIG. 98). Then, the red PDPA-TMRsignals started to emerge at 20-30 min. All the red dots werecolocalized with a subset of green dots at this stage. Finally, thePDBA-Cy5 component was activated with pseudocolored blue dots at 90-180min, and all the blue dots were colocalized with a subset of red dots atthis period. The endocytic organelles can be divided into threepopulations: (i) green dots (6.2<pH<6.9); (ii) yellow dots (5.3<pH<6.2);(iii) white dots (pH<5.3), which indicated pH-6.8 for clathrin-coatedvesicle, pH-6.0 for early endosome and pH 5.0-5.5 for lateendosome/lysosome, respectively. Similarly, the sequential activationwas observed inside the single acidic organelle in HN5 head-neck cancercell line (FIG. 99). Thus, the hybrid UPS nanoprobe successfullyreported spatiotemporal pH changes along the specific endocytic pathwayin single organelle resolution.

Having demonstrated the unique capability of the hybrid UPS nanoprobe,the unique oncogenic signature that is responsible for the dramaticincrease of acidification rates during organelle maturation wasinvestigated. Seven lung cancer cell lines with different gene mutationbackground were selected and evaluated (Table 15). The cells wereincubated with 100 ag/mL Erb-encoded hybrid UPS nanoprobe at 4° C. for30 min, washed three times and then imaged in real time at 37° C. totrack the nanoprobe activation rates which indicates the organelleacidification capacity (FIGS. 100-102). Results showed that theactivation rates of KRAS mutated cells including HCC44, H2009, H460, andA549 are significantly faster than KRAS wild type cells (H2882, H1991,and H1819). To normalize the uptake difference in different cell lines,the fluorescence intensity of PDPA-TMR (early endosome) and PDBA-Cy5(late endosome/lysosome) signals were divided by PEPA-BDY493 signal,which was activated at as early as 15 min. The I_(6.2)/I_(6.9) andI_(5.3)/I_(6.9) as a function of time were plotted (FIGS. 103A & 103Band FIGS. 104A-104D). At 30 min, no significant activation difference ofTMR channel (I_(6.2)/I_(6.9)) was observed. At 75 min, theI_(5.3)/I_(6.9) ratios for KRAS mutated cells reached to 70%blue-positive organelles, while KRAS wild type cells only had less than40% blue-positive organelles. These results indicated that KRAS mutationwould be responsible for the lysosome catabolism associated with pHregulation.

TABLE 15 Cell lines and their gene mutation background Cell line KRASstatus P53 status HCC44 Mutate (M) M H2009 M M H460 M Wild Type (WT)A549 M WT H2882 WT M H1993 WT M H1819 WT WT

Given that the KRAS mutaton is probably responsible for the upregulatedacidification rate of lysosome, the hybrid UPS nanoprobe was utilized todirectly capture the organelle pH correlates with gene mutations. As amodel system, tumor-derived (HCC4017) and normal bronchioleepithelia-derived (HBEC30KT) cell lines from the same lung cancerpatient together with an isogenic progression series of HBEC30KT withstepwise stable suppression of TP53 (HBEC30KT-shTP53), stable expressionof KRAS^(G12V) (HBEC30KT-shTP53/KRAS^(G2V)), and stable suppression ofLKB1 (HBEC30KT-shTP53/KRAS^(G12V)/shLKB1) were selected and imaged(Ramirez et al., 2004). FIGS. 105A & 105B showed the dramatic differenceon the faster maturation rates of endocytic organelles in malignantHCC4017 cells over HBEC30KT epithelial cells (FIGS. 106 & 107).Genotyping of the parental tumor and HCC4017 cell line revealedmutations in TP53, LKB1 and KRAS. The data is consistent with previousfindings that KRAS/LKB1 mutated cells rely on lysosomal catabolism forgrowth and survival (Kim et al., 2013). To further pinpoint whichoncogenic signature is responsible for the difference in organellematuration, the fluorescence activation pattern of hybrid nanoprobe inthe isogenic progression series of HBEC30KT were imaged (FIGS. 108-110).The I_(6.2)/I_(6.9) and I_(5.3)/I_(6.9) as a function of time wereplotted (FIGS. 111A-111D). Results clearly indicated that KRAS mutationis responsible for the dramatic increase in the acidification andmaturation of endocytic organelles. 2, Method and Materials

1. Synthesis and Characterization of Hybrid Nanoprobes

Dye conjugated PEG-b-PR and maeimide-terminated PEG-b-PDPA(Mal-PEG-PDPA) block copolymers were synthesized by the atom transferradical polymerization method. The hybrid nanoprobes were preparedfollowing a previously published procedure (Wang et al., 2014). In atypical procedure, 5 mg of each PEG-b-PEPA-BDY493, PEG-b-PDPA-TMR, andPEG-b-PDBA-Cy5 polymer were dissolved in 1 mL THF. Then, the mixture wasadded into 10 mL of Milli-Q water under sonication. The mixture wasfiltered four times to remove THF using a micro-ultracentrifugationsystem. Then, the distilled water was added to adjust the final polymerconcentration to 5 mg/mL. To prepare the Erbitux-conjugate hybridnanoprobe, 0.6 mg Mal-PEG-PDPA, 4 mg of each PEG-b-PEPA-BDY493,PEG-b-PDPA-TMR, and PEG-b-PDBA-Cy5 polymer were dissolved in 1 mL THF,and the same procedure as described above was used to prepare the 5%maleimide-modified hybrid nanoprobe. Meanwhile, the Erbitux Fab′-SHfragment (3 mg, M_(w)=55 kDa) was prepared following the publishedprocedure. Then, the maleimide-modified hybrid nanoprobe and ErbituxFab′-SH solution were mixed and reacted in 100 mM phosphate bufferedsaline (PBS, pH 7.4) containing 1 mM EDTA overnight at room temperature.Then, the mixture was filtered six times to remove free Fab′-SH using amicro-ultracentrifugation system (MWCO=100K, Millipore). Then, 100 mMPBS (pH 7.4) was added to adjust the final polymer conentration to 5mg/mL. Transmission electron microscopy was carried out with 1%phosphotungstic acid negative staining and visualized on a JEOL 1200EXelectron microscope (JEOL 1200EX). The particle size and distribution ofthe nanoparticles were determined by dynamic light scattering (DLS)analysis. The mean count rates of the nanoparticles as a function of pHvalues were also determined by DLS analysis.

2. Fluorescence Activation of UPS Nanoprobes

Fluorescence emission spectra of the hybrid UPS nanoprobes in differentpH buffer solutions were obtained on a Hitachi fluorometer (F-7500model). The final polymer concentration was adjusted to 100 ag/mL using100 mM PBS with different pH values. The hybrid nanoprobe was excited at485, 545, and 640 nm, respectively. The corresponding emission spectrawere collected at 490-750, 560-750, and 650-750 nm, respectively. Theemission peaks at 510, 580, and 710 nm were used to quantify thefluorescence activation ratios for BDY493, TMR, and Cy5 channels.Fluorescent images of the hybrid nanoprobe solution (100 μg/mL) atdifferent pH values were captured on a Maestro imaging system (CRI)using blue (515 nm LP), green (580 nm LP), and orange (645 nm LP)filters. Then, the images were spectrally unmixed using the standardfluorescent spectra of individual dyes to obtain the multicolor images.

3. Cell Culture

The lung cancer cell line A549 and head and neck cancer cell line HN5were culture in DMEM (Invitrogen) containing 10% fetal bovine serum(Invitrogen), 100 IU/mL penicillin, and 100 μg/mL streptomycin(Invitrogen). The HBEC30KT progression series and HCC4017 cells werecultured in ACL4 medium supplemented with 2% fetal bovine serum andantibiotics at 37° C. in 5% CO₂ atmosphere.

4. Cell Imaging

A549 and HN5 cells were plated in glass bottom dishes (MatTek, MA) in 2mL complete DMEM medium. To test the specificity of Erbitux-conjugatednanoprobes, the A549 cells in complete medium were kept at 4° C. for 10min, then 100 μg/mL of Erb-PDPA-TMR micelle was added and kept for 30min at 4° C. for epidermal growth factor receptor (EGFR) binding, thenthe medium was removed and washed with ice-cold PBS three times.Thereafter, cells were incubated with complete medium for 2 h at 37° C.The confocal images were captured by a Nikon ECLIPSE TE2000-E confocalmicroscope with a 60× objective lens.

To demonstrate the synchronized uptake of three components in the hybridnanoprobe, the A549 cells in complete medium were kept at 4° C. for 10min, then 100 μg/mL of Erb-conjugated hybrid nanoprobe was added andkept for 30 min at 4° C. for EGFR binding. The medium was removed andwashed thrice. Thereafter, cells were incubated with complete medium for3 h at 37° C. BDY493, TMR, and Cy5 were excited at 488, 543, and 633 nm,respectively. The FITC (515±15 nm), TRITC (590±25 nm) and Cy5 (650 nmLP) filters were used for BDY493, TMR, and Cy5 imaging, respectively.For control group, PEPA-BDY493, Erb-PDPA-TMR, and PDBA-Cy5 micelles wereprepared, mixed up, and incubated with A549 cells. The same procedurewas utilized for the cell imaging.

To track endosome maturation process, the cell samples were preparedusing the same procedure described above. Confocal images were capturedby a Nikon ECLIPSE TE2000-E confocal microscope with a 100× objectivelens at 0, 15, 30, 60 min, 2.5, and 5 hrs after incubation at 37° C. TheBDY493, TMR, and Cy5 three channels were excited and collected using thesame setting described above. The images were analyzed using Image-Jsoftware.

Five independent measurements were presented as the mean±standarddeviation.

5. Statistical Analysis

Data were expressed as mean±s.d. Differences between groups wereassessed using paired, two-sided Student t-test. *P<0.05 was consideredsignificant, and **P<0.01 was considered highly significant.

6. Materials

Tetramethylrhodamine succinimidyl ester (TMR-NHS) and BODIPY®493/503succinimidyl ester (BDY493-NHS) were purchased from Invitrogen Inc. Cy5NHS ester (Cy5-NHS) was purchased from Lumiprobe Company. Monomersincluding 2-(dipropylamino) ethyl methacrylate (DPA-MA), and2-(dibutylamino) ethyl methacrylate (DBA-MA) were reported recently(Zhou et al., 2011; Ma et al., 2014). 2-aminoethyl methacrylate (AMA)was purchased from Polyscience Company. AMA was recrystallized twicewith isopropanol and ethyl acetate (3:7). PEG macroinitiator,MeO-PEG₁₁₄-Br, was prepared from α-bromoisobutyryl bromide andMeO-PEG₁₁₄-OH according to the procedure in literature (Zhou et al.,2011). Other solvents and reagents were used as received fromSigma-Aldrich or Fisher Scientific Inc.

7. Synthesis of PEG-b-(PR-r-Dye) Block Copolymers

PEG-b-(PR-r-AMA) copolymers (Scheme 1) were first synthesized by atomtransfer radical polymerization (ATRP) method. The primary amino groupswere introduced into each polymer chain by controlling the feeding ratioof the AMA monomer to the initiator (ratio=3). The dye-free copolymerswere used in polymer characterizations (Table 12). PEG-b-P(DPA-r-AMA)was used as an example to illustrate the procedure. First, DPA-MA (1.7g, 8 mmol), AMA (50 mg, 3 mmol), PMDETA (21 μL, 0.1 mmol), andMeO-PEG₁₁₄-Br (0.5 g, 0.1 mmol) were charged into a polymerization tube.Then a mixture of 2-propanol (2 mL) and DMF (2 mL) was added to dissolvethe monomer and initiator. After three cycles of freeze-pump-thaw toremove oxygen, CuBr (14.4 mg, 0.1 mmol) was added into the reaction tubeunder nitrogen atmosphere, and the tube was sealed in vacuo. Thepolymerization was carried out at 40° C. for 12 hours. Afterpolymerization, the reaction mixture was diluted with 10 mL THF, andpassed through an Al₂O₃ column to remove the catalyst. The THF solventwas removed by rotovap. The residue was dialyzed in distilled water andlyophilized to obtain a white powder. The resulting PEG-b-(PR-r-AMA)copolymers were characterized by 500 MHz ¹H-NMR, gel permeationchromatography (Viscotech GPCmax, PLgel 5 μm MIXED-D columns by PolymerLabs, THF as eluent at 1 mL/min). Table 12 enlists the yield, molecularweights (M_(n) and M_(w)) and polydispersity index (PDI) of eachcopolymer.

Synthesis of dye-conjugated copolymers followed a representativeprocedure described below. For TMR conjugation, PEG-b-P(DPA-r-AMA) (50mg) was first dissolved in 2 mL DMF. Then, TMR-NHS ester (1.5equivalents to the molar amount of the primary amino group) was added.The reaction mixture was stirred at room temperature for 24 hours. Thecopolymers were purified by preparative gel permeation chromatography(PLgel Prep 10 μm 10E3{acute over (Å)} 300×25 mm columns by Varian, THFas eluent at 5 mL/min) to remove the free dye molecules. The producedPEG-b-P(DPA-TMR) copolymers were lyophilized and stored at −20° C. forfurther research. The dye conjugation efficiency and quantum yield weredetermined according to the procedure in the literature (Ma et al.,2014).

8. Preparation of the Micelle Nanoparticles

Micelles were prepared following a previously published procedure. In atypical procedure, 10 mg of PDPA-TMR was dissolved in 0.5 mL THF. Then,the mixture was slowly added into 4 mL of Milli-Q water undersonication. The mixture was filtered 4 times to remove THF using themicro-ultrafiltration system. Then, the distilled water was added toadjust the polymer concentration to 5 mg/mL as a stock solution. For themulti-color hybrid nanoparticle, 5 mg of PEPA-BDY, 5 mg of PDPA-TMR, and5 mg of PDBA-Cy5 were dissolved in 1 mL THF. Then, the same procedurewas used to prepare the hybrid nanoparticle. The nanoparticles werecharacterized by transmission electron microscopy (TEM, JEOL 1200 EXmodel) for micelle size and morphology, dynamic light scattering (DLS,Malvern Zetasizer Nano-ZS, λ=632 nm) for hydrodynamic diameter (D_(h)).

For Erbitux-conjugated hybrid nanoprobe, 4 mg of PEPA-BDY, 4 mg ofPDPA-TMR, 4 mg of PDBA-Cy5, and 0.6 mg of MAL-PEG-PDPA were dissolved in1 mL THF. Then, the same procedure was used to prepare the Mal-hybridnanoprobe. After micelle formation, an excess amount of Erbitux Fab′-SHfragment (55 kD) in PBS buffer containing 1 mM EDTA was added. Theconjugation was allowed to occur overnight under N₂ atmosphere followedby ultracentrifugation six times to remove free Fab′-SH. The resultingErb-conjugated hybrid nanoprobe was adjusted to 5 mg/mL polymerconcentration for cell imaging studies. The Erb-conjugated PDPA-TMRmicelle was also prepared using the same procedure.

9. Fluorescence Characterization

The fluorescence emission spectra in different pH buffer solutions wereobtained on a Hitachi fluorometer (F-7500 model). For each polymericmicelle, the sample (5 mg/mL) was prepared in Milli-Q water. Then, thesolution was diluted in 100 mM phosphate buffered saline (PBS) withdifferent pH values. The final polymer concentration was controlled at0.1 mg/mL.

To demonstrate whether different polymer can form a homogenous hybridmicelle, we examined the fluorescence properties of hybrid micellesusing fluorescence resonance energy transfer (FRET) experiments. Foreach nanoprobe, the sample (5 mg/mL) was prepared in Milli-Q water. Thesolution was diluted to 100 μg/mL in 100 mM PBS buffer (pH 7.4). Then,the nanoprobe was excited by a proper excitation light (λ_(ex)=485, 545,and 640 nm), and the emission spectra were collected.

The fluorescent images of hybrid nanoprobe solutions (0.1 mg/mL) atdifferent pH were captured on Maestro in vivo imaging system (CRI Inc.Woburn, Mass.) using a proper band pass excitation filter and a properlong-pass emission filter.

10. Cell Culture

Human lung small cell lung cancer A549 cells and head and neck cancerHN5 cells were cultured in DMEM medium (Invitrogen, CA) supplementedwith 10% fetal bovine serum (FBS), 100 IU/mL penicillin and 100 jag/mLstreptomycin at 37° C. in 5% CO₂ atmosphere.

Tumor-derived (HCC4017) and normal bronchiole epithelia-derived (HBEC30)cell lines from the same lung cancer patient were obtained. The normalbronchial epithelial cells were immortalized by stable expression ofCDK4 and hTERT to produce HBEC30KT. Series cell lines of HBEC30KTderivatives with stepwise stable suppression of p53 (HBEC30KT-shTP53),stable expression of KRAS^(G12V) (HBEC30KT-shTP53/KRAS^(G12V)), andstable suppression of LKB1 (HBEC30KT-shTP53/KRASG^(12V)/shLKB1) werealso obtained.

The HBEC30KT progression series and HCC4017 cells were cultured in ACL4medium supplemented with 2% fetal bovine serum (FBS), 100 IU/mLpenicillin and 100 ag/mL streptomycin at 37° C. in 5% CO₂ atmosphere.

11. Multi-Stage Activation of Erbitux-Conjugated Hybrid Nanoprobes inLiving Cells

A549 and HN5 Cells were plated in glass bottom dishes (MatTek, MA) in 2mL complete DMEM medium. To test the specificity of Erbitux-conjugatednanoprobes, the A549 cells were incubated with complete mediumcontaining Erb-PDPA-TMR micelle for 1 hour at 37° C., then the mediumwas removed and washed 3 times. The confocal images were captured by aNikon ECLIPSE TE2000-E confocal microscope with a 60× objective lens.

To demonstrate the synchronized uptake of three components in the hybridnanoprobe, the A549 cells in complete medium were kept at 4° C. for 10min, then 100 μg/mL of Erb-conjugated hybrid nanoprobe was added andkept for 30 min at 4° C. for epidermal growth factor receptor (EGFR)binding. The medium was removed and washed with ice-cold PBS threetimes. Thereafter, cells were incubated with complete medium for 3 hoursat 37° C. BDY493, TMR, and Cy5 were excited at 488, 543, and 633 nm,respectively. The FITC (515±15 nm), TRITC (590±25 nm) and Cy5 (650 nmLP) filters were used for BDY493, TMR, and Cy5 imaging, respectively.For control group, PEPA-BDY493, Erb-PDPA-TMR, and PDBA-Cy5 micelles wereprepared, mixed up, and incubated with A549 cells. The same procedurewas utilized for the pulse-chase study.

12. Tracking Endosome Maturation During Endocytosis UsingErbitux-Conjugated Hybrid Nanoprobes

Pulse chase experiments were utilized to track endosome maturationprocess. Cells in complete medium were kept at 4° C. for 10 min, andthen 100 μg/mL of Erb-conjugated hybrid nanoprobe was added and kept for30 min at 4° C. for EGFR binding. The medium was removed and washed withice-cold PBS three times. Thereafter, cells were incubated with completemedium at 37° C. Confocal images were captured by a Nikon ECLIPSETE2000-E confocal microscope with a 100× objective lens at 0, 15, 30, 60min, 2.5, and 5 hrs after addition of micelles. BDY493, TMR, and Cy5were excited at 488, 543, and 633 nm, respectively. The FITC (515±15nm), TRITC (590±25 nm) and Cy5 (650 nm LP) filters were used for BDY493,TMR, and Cy5 imaging, respectively. The images were analyzed usingImage-J software. Five independent measurements were presented as themean±standard deviation.

Example 15: Syntheses of Triblock Copolymer PEO-b-P(R₁-b-R₂)

PEG-b-P(R₁-b-R₂) triblock copolymers were synthesized by ATRP methodfollowing similar procedures previously reported. PEO-b-P(DSA-b-DEA) isused as an example to illustrate the procedure. First, D5A-MA (0.54 g, 2mmol), PMDETA (12 μL, 0.05 mmol) and MeO-PEO₁₁₄-Br (0.25 g, 0.05 mmol)were charged into a polymerization tube. Then a mixture of 2-propanol (1mL) and DMF (1 mL) was added to dissolve the monomer and initiator.After three cycles of freeze-pump-thaw to remove the oxygen, CuBr (7 mg,0.05 mmol) was added into the polymerization tube under nitrogenatmosphere, and the tube was sealed in vacuo. After polymerizationcarrying out at 40° C. for 8 hours, deoxygenized DEA-MA (0.368, 2 mmol)was injected to the reaction solution via air-tight syringe and thereaction mixture was stirred at 40° C. for additional 8 hours. Afterpolymerization, the reaction mixture was diluted with 10 mL THF, andpassed through a neutral Al₂O₃ column to remove the catalyst. The THFsolvent was removed by rotovap. The residue was dialyzed in distilledwater and lyophilized to obtain a white powder. PEO-b-P(DEA-b-DSA) canalso be synthesized by reversing the feeding sequence of DEA and D5A.The pH titration experiments showed two distinctive ionizationtransitions for the PEO-b-P(DSA₄₀-b-DEA₄₀) or PEO-b-P(DEA₄₀-b-D5A₄₀). Incontrast, only one pH transition was observed for the correspondingrandom PR block copolymers (FIGS. 113A & 113B).

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this disclosure havebeen described in terms of certain embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe methods described herein without departing from the concept, spiritand scope of the disclosure. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the disclosure as defined by theappended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

-   WO 2013/152059-   Adams & Weiner, Nat Biotechnol, 23:1147-1157, 2005.-   Albertazzi, L.; Storti, B.; Marchetti, L.; Beltram, F. J. Am. Chem.    Soc. 2010, 132, 18158.-   Alberts, B.; Johnson, A.; Lewis, J.; Raff, M.; Roberts, K.;    Walter, P. Molecular Biology of the Cell; 5th ed.; Garland Science:    New York, 2008.-   Almutairi, A.; Guillaudeu, S. J.; Berezin, M. Y.; Achilefu, S.;    Frechet, J. M. J. J. Am. Chem. Soc. 2007, 130, 444.-   Ananthapadmanabhan, K. P.; Goddard, E. D.; Turro, N. J.; Kuo, P. L.    Langmuir 1985, 1, 352.-   Atkins, et al., J. Surg. Res., 177, 109-115, 2012.-   Atkins, P.; De Paula, J. Physical Chemistry; Oxford University    Press, 2009.-   Benjaminsen, R. V.; Sun, H. H.; Henriksen, J. R.; Christensen, N.    M.; Almdal, K.; Andresen, T. L. ACS Nano 2011, 5, 5864.-   Berezin, M. Y.; Achilefu, S. Chem. Rev. 2010, 110, 2641.-   Blum, G.; Mullins, S. R.; Keren, K.; Fonovic, M.; Jedeszko, C.;    Rice, M. J.; Sloane, B. F.; Bogyo, M. Nat. Chem. Biol. 2005, 1, 203.-   Bolte & Cordelieres, J. Microsc. 224, 213-232, 2006.-   Cardone et al., Nat. Rev. Cancer 5, 786-795, 2005.-   Casey et al., Nat. Rev. Mol. Cell Biol., 11:50-61, 2010.-   Choi et al., Nat. Biotechnol. 31, 148-153, 2013.-   Christofk et al., Nature, 452:230-233, 2008.-   Collins and Washabaugh, Q. Rev. Biophys. 18:323-422, 1985.-   Collins, Biophys. J. 1997, 72, 65-76.-   Conner & Schmid, Nature, 422:37-44, 2003.-   Cook et al., Semin. Nucl. Med. 34, 122-133, 2004.-   Cross & Muller, FEBS Lett., 576:1-4, 2004.-   Dacosta et al., Best Pract. Res. Clin. Gastroenterol., 20, 41-57,    2006.-   Dai, S.; Ravi, P.; Tam, K. C. Soft Matter 2008, 4, 435.-   Dale, T. J.; Rebek, J. J. Am. Chem. Soc. 2006, 128, 4500.-   de Silva, A. P.; Gunaratne, H. Q. N.; Gunnlaugsson, T.;    Huxley, A. J. M.; McCoy, C.; P.; Rademacher, J. T.; Rice, T. E.    Chem. Rev. 1997, 97, 1515.-   de Silva, A. P.; Gunaratne, H. Q. N.; McCoy, C. P. Chem. Commun.    1996, 2399.-   Deamer et al., Membrane permeability: 100 years since Ernest    Overton. (Academic Press, San Diego, Calif., USA; 1999).-   Demaurex, N. News Physiol. Sci. 2002, 17, 1-5.-   Demchenko, A. P. Introduction to Fluorescence Sensing; Springer    Science: New York, 2008.-   Diaz-Fernandez et al., Chemistry 12, 921-930, 2006.-   Diaz-Fernandez, Y.; Foti, F.; Mangano, C.; Pallavicini, P.; Patroni,    S.; Perez-Gramatges, A.; Rodriguez-Calvo, S. Chem. Eur. J. 2006, 12,    921.-   Diwu et al., Chem. Biol., 6: 411-418, 1999.-   Draga et al., Anal. Chem., 82, 5993-5999, 2010.-   Eigenbrodt et al., Anticancer Res., 18:3267-3274, 1998.-   Enerson & Drewes, J. Pharm. Sci., 92:1531-1544, 2003.-   Fantin et al., Cancer Cell, 9:425-434, 2006.-   Fernandez-Suarez, M.; Ting, A. Y. Nat. Rev. Mol. Cell Biol. 2008, 9,    929.-   Folkman, J., Nat. Rev. Drug Discovery, 2007, 6, 273-286.-   Fukui et al., Radiographics 25, 913-930, 2005.-   Gallagher et al., Nature 453, 940-943, 2008.-   Gatenby, R. A.; Gillies, R. J., Nat. Rev. Cancer, 2004, 4, 891-899.-   Gatenby, R. A.; Gillies, R. J., Nat. Rev. Cancer, 2008, 8, 56-61.-   Giepmans, B. N. G.; Adams, S. R.; Ellisman, M. H.; Tsien, R. Y.    Science 2006, 312, 217.-   Gil, E. S.; Hudson, S. M. Prog. Polym. Sci. 2004, 29, 1173.-   Gillies et al., Am. J. Physiol., 267, C195-C203, 1994.-   Gillies et al., Med. Biol. Mag., 23, 57-64, 2004.-   Gross, S.; Piwnica-Worms, D. Cancer Cell 2005, 7, 5.-   Grunwell, et al., J. Am. Chem. Soc. 2001, 123, 4295-4303.-   Ha, et al., Proc. Natl. Acad. Sci. U.S.A 1999, 96, 893-898-   Haka et al., Cancer Res., 66, 3317-3322, 2006.-   Halestrap & Price, Biochem. J., 343 Pt 2, 281-299, 1999.-   Han, J. Y.; Burgess, K. Chem. Rev. 2010, 110, 2709.-   Hanahan, D.; Weinberg, R. A., Cell, 2011, 144, 646-674.-   Haubner & Wester, Curr Pharm Des., 10:1439-1455, 2004.-   Hawkins & Davis, Pharmacological Reviews, 57:173-185, 2005.-   Heiden et al., Science, 324, 1029-1033, 2009.-   Hofmeister, Pathol. u. Pharmakol, 25, 1888.-   Holtzman, E. Lysosomes. (Springer, New York, N.Y., USA), 1989.-   Huang, X. et. al., ACS Nano, 2010, 4, 5887-5896.-   Huotari & Helenius, EMBO J, 30:3481-3500, 2011.-   Iczkowski and Lucia, Prostate Cancer, 2011, 673021, 2011.-   Imamura et al., Proc. Natl. Acad. Sci. U.S.A, 100: 2312-2315, 2003.-   Izumi et al., Cancer Treat. Rev. 2003, 29, 541.-   Jares-Erijman and Jovin, Nat Biotech 2003, 21, 1387-1395.-   Jelley, E. E. Nature 1936, 138, 1009.-   Johansson, M. K.; Cook, R. M. Chem. Eur. J. 2003, 9, 3466.-   Jose et al., Biochim. Biophys. Acta, 1807:552-561, 2011.-   Joyce, J. A., Cancer Cell, 2005, 7, 513-520.-   Kairdolf, B. A.; Nie, S. J. Am. Chem. Soc. 2011, 133, 7268.-   Kalyanasundaram & Thomas, J. Am. Chem. Soc., 99:2039-2044, 1977.-   Kanter et al., Am. J. Obstet. Gynecol., 200, 512 e511-515, 2009.-   Kim et al., Cell, 155:552-566, 2013.-   Kleiter, M. M. et. al., Int. J. Radiat. Oncol. Biol. Phys., 2006,    64, 592-602.-   Kobayashi, H.; Choyke, P. L. Acc. Chem. Res. 2010, 44, 83.-   Kobayashi, H.; Ogawa, M.; Alford, R.; Choyke, P. L.; Urano, Y. Chem.    Rev. 2010, 110, 2620.-   Kreuter, Advanced Drug Delivery Reviews, 47:65-81, 2001.-   Kubben et al., Lancet Oncol., 12, 1062-1070, 2011.-   Kunz, et al., Curr. Opin. Colloid Interface Sci. 2004, 9, 1-18.-   Lakowicz, J. R. Principles of Fluorescence Spectroscopy; 3rd ed.;    Springer: New York City, 2006.-   Lee, E. S.; Shin, H. J.; Na, K.; Bae, Y. H. J. Controlled Release    2003, 90, 363.-   Lee, S.; Park, K.; Kim, K.; Choi, K.; Kwon, I. C. Chem. Commun.    2008, 4250.-   Lee, S.; Ryu, J. H.; Park, K.; Lee, A.; Lee, S. Y.; Youn, I. C.;    Ahn, C. H.; Yoon, S. M.; Myung, S. J.; Moon, D. H.; Chen, X.; Choi,    K.; Kwon, I. C.; Kim, K. Nano Lett. 2009, 9, 4412.-   Lee, S.; Xie, J.; Chen, X. Y. Curr. Top. Med. Chem. 2010, 10, 1135.-   Leontidis, Curr. Opin. Colloid Interface Sci. 2002, 7, 81-91.-   Levi, J.; Kothapalli, S. R.; Ma, T. J.; Hartman, K.; Khuri-Yakub, B.    T.; Gambhir, S. S. J. Am. Chem. Soc. 2010, 132, 11264.-   Li et al., Angew. Chem. Int. Ed. Engl., 53:8074-8078, 2014.-   Li, C.; Xia, J. A.; Wei, X. B.; Yan, H. H.; Si, Z.; Ju, S. H. Adv.    Funct. Mater. 2010, 20, 2222.-   Liu et al., ACS Nano, 4, 2755-2765, 2010.-   Liu et al., Angew Chem Int Ed Engl., 48:7346-7349, 2009.-   Lodder et al., Eur. Radiol., 21, 98-106, 2011.-   López Arbeloa, I.; Ruiz Ojeda, P. Chem. Phys. Lett. 1982, 87, 556.-   Lou et al., Cancer Res. 71, 3364-3376, 2011.-   Lovell, J. F.; Liu, T. W. B.; Chen, J.; Zheng, G. Chem. Rev. 2010,    110, 2839.-   Ma, et al., J. Am. Chem. Soc., 136:11085-11092, 2014.-   Ma, et al., Macromolecules 2003, 36, 3475.-   Manders et al., J. Microsc., 169: 375-382, 1993.-   Maxfield & McGraw, Nat. Rev. Mol. Cell Biol. 5, 121-132, 2004.-   Maxfield, F. R.; McGraw, T. E. Nat. Rev. Mol. Cell Biol. 2004, 5,    121.-   Maxwell, D.; Chang, Q.; Zhang, X.; Barnett, E. M.; Piwnica-Worms, D.    Bioconjug. Chem. 2009, 20, 702.-   McElroy et al., World J. Surg., 32, 1057-1066, 2008.-   McMahon et al., Br. J. Oral Maxillofac. Surg., 41, 224-231, 2003.-   Merk, et al., Langmuir 2014, 30, 4213-4222.-   Mirbolooki, EJNMMI Research, 1:30, 2011.-   Mo et al., Anal. Chem., 81, 8908-8915, 2009.-   Modi, S.; Swetha, M. G.; Goswami, D.; Gupta, G. D.; Mayor, S.;    Krishnan, Y. Nat. Nanotech. 2009, 4, 325.-   Morgan, Mol. Membr. Biol., 21:423-433, 2004.-   Nasongkla, N.; Bey, E.; Ren, J. M.; Ai, H.; Khemtong, C.; Guthi, J.    S.; Chin, S. F.; Sherry, A. D.; Boothman, D. A.; Gao, J. M., Nano.    Lett. 2006, 6, 2427-2430.-   Neri & Supuran, Nat. Rev. Drug Discov. 10, 767-777, 2011.-   Neri & Supuran, Nat. Rev. Drug Discov., 10:767-777, 2011.-   Nguyen and Tsien, Nat. Rev. Cancer, 13, 653-662, 2013.-   Nishi, T.; Forgac, M. Nat. Rev. Mol. Cell Biol. 2002, 3, 94.-   Ogawa, M.; Kosaka, N.; Choyke, P. L.; Kobayashi, H. ACS Chem. Biol.    2009, 4, 535.-   Ohgaki, R.; van Ijzendoorn, S. C. D.; Matsushita, M.; Hoekstra, D.;    Kanazawa, H. Biochemistry 2010, 50, 443.-   Pacchiano et al., J. Med. Chem. 54, 1896-1902, 2011.-   Packard, B. Z.; Komoriya, A.; Toptygin, D. D.; Brand, L. J. Phys.    Chem. B 1997, 101, 5070.-   Parks et al., Nat. Rev. Cancer 13, 611-623, 2013.-   Parsons, et al., Phys. Chem. Chem. Phys. 2011, 13, 12352-12367.-   Pena-Llopis et al., EMBO J., 30: 3242-3258, 2011.-   Perez-Sayans et al., Cancer Treat. Rev., 35:707-713, 2009.-   Petsalakis, I. D.; Lathiotakis, N. N.; Theodorakopoulos, G. J. Mol.    Struct.: THEOCHEM 2008, 867, 64.-   Pouyssegur et al., Nature, 441:437-443, 2006.-   Ramanujam et al., Photochem. Photobiol, 64, 720-735, 1996.-   Ramirez et al., Cancer Res, 64: 9027-9034, 2004.-   Ravasz et al., J. Craniomaxillofac. Surg., 19, 314-318, 1991.-   Riess, G. Prog. Polym. Sci. 2003, 28, 1107.-   Roczniak-Ferguson et al., Sci Signal 5, ra42, 2012.-   Rodman et al., Exp. Cell Res., 192:445-452, 1991.-   Rossin et al., J Nucl Med., 49:103-111, 2008.-   Ruckenstein, E.; Nagarajan, R. J. Phys. Chem. 1975, 79, 2622.-   Sancak et al., Cell 141: 290-303, 2010.-   Sapsford, et al., Angew. Chem. Int. Ed. 2006, 45, 4562-4589.-   Scheibe, G. Z. Angew. Chem. 1936, 49, 563.-   Schomacker et al., Lasers Surg. Med., 12, 63-78, 1992.-   Schwarz et al., Cancer, 115, 1669-1679, 2009.-   Settembre et al., EMBO J., 31:1095-1108, 2012.-   Settembre et al., Nat. Rev. Mol. Cell Biol., 14:283-296, 2013.-   Sonveaux et al., J. Clin. Invest. 118, 3930-3942, 2008.-   Srikun, D.; Albers, A. E.; Chang, C. J. Chem. Sci. 2011, 2, 1156.-   Sterling, et al., Cell Physiology, 283:C1522-1529, 2002.-   Suh et al., Bioorg. Chem., 22: 318-327, 1994.-   Sun et al., J Med Chem., 45:469-477, 2002.-   Supuran, Bioorg. Med. Chem. Lett., 20:3467-3474, 2010.-   Supuran, Nat. Rev. Drug Discov., 7:168-181, 2008.-   Swartz, M. A.; et. al.; Cancer Res., 2012, 72, 2473-2480.-   Tal, S.; Salman, H.; Abraham, Y.; Botoshansky, M.; Eichen, Y. Chem.    Eur. J. 2006, 12, 4858.-   Tran et al., Hepatogastroenterology, 59, 1994-1999, 2012.-   Tsarevsky and Matyjaszewski, Chem. Rev. 2007, 107, 2270.-   Tsien, R. Y. Nat. Rev. Mol. Cell Biol. 2003, 4, SS16.-   Ueno, T.; Nagano, T. Nat. Methods 2011, 8, 642.-   Underwood and Anacker, J. Colloid Interface Sci. 1987, 117, 242-250.-   Urano, et al., Nat. Med., 15:104-109, 2008.-   Urano, Y.; Asanuma, D.; Hama, Y.; Koyama, Y.; Barrett, T.; Kamiya,    M.; Nagano, T.; Watanabe, T.; Hasegawa, A.; Choyke, P. L.;    Kobayashi, H. Nat. Med. 2009, 15, 104.-   Vahrmeijer et al., Nat. Rev. Clin. Oncol., 10, 507-518, 2013.-   Valdes-Aguilera, O.; Neckers, D. C. Acc. Chem. Res. 1989, 22, 171.-   Valeur, B. Molecular fluorescence: principles and applications;    Wiley-VCH, 2002.-   van Sluis et al., Magn. Reson. Med., 41, 743-750, 1999.-   Vishvakarma & Singh, Biomed. Pharmacother. 65, 27-39, 2011.-   Vogel, S. S.; Thaler, C.; Koushik, S. V. Sci. STKE 2006, 2006, re2.-   Volk et al., Br. J. Cancer, 68, 492-500, 1993.-   Wadas et al., Curr Pharm Des. 13:3-16, 2007.-   Wang et al., J Vis Exp., 2012.-   Wang et al., Journal of Visualized Experiments:JoVE, 2012.-   Wang et al., Nat. Mater., 13:204-212, 2014.-   Wang, et al., Angew. Chem. Int. Ed. 2008, 47, 9726-9729.-   Wasielewski, M. R. Chem. Rev. 1992, 92, 435.-   Webb et al., Nat. Rev. Cancer, 11, 671-677, 2011.-   Webb, B. A.; Chimenti, M.; Jacobson, M. P.; Barber, D. L., Nat. Rev.    Cancer, 2011, 11, 671-677.-   Weerakkody et al., Proc. Natl. Acad. Sci. U.S.A 110, 5834-5839,    2013.-   Weis, S. M., Cheresh, D. A., Nat. Med., 2011, 17, 1359-1370.-   Weissleder, R.; Pittet, M. J. Nature 2008, 452, 580.-   Weisz, Cytol., 226:259-319, 2003.-   Weller, A. Pure Appl. Chem. 1968, 16, 115.-   West, W.; Pearce, S. J. Phys. Chem. 1965, 69, 1894.-   Winnik, Chem. Rev., 93:587-614, 1993.-   Woodin et al., Eur J Inorg Chem., 4829-4833, 2005.-   Woolgar and Triantafyllou, Oral Oncol., 41, 1034-1043, 2005.-   Yang C. et. al., Cancer Res. 2009, 69, 7986-93.-   Yeung et al., Curr. Opin. Cell Biol. 18, 429-437, 2006.-   Yezhelyev et al., J. Am. Chem. Soc. 2008, 130, 9006.-   Yu et al., ACS Nano 2011, 5, 9246.-   Zhang and Cremer, Curr. Opin. Chem. Biol. 2006, 10, 658-663.-   Zhang and Cremer, Proc. Natl. Acad. Sci. U.S.A 2009, 106,    15249-15253.-   Zhang et al., Cell Metab. 20:526-540, 2014.-   Zhang, J.; Campbell, R. E.; Ting, A. Y.; Tsien, R. Y. Nat. Rev. Mol.    Cell Biol. 2002, 3, 906.-   Zhang, X.; Lin, Y.; Gillies, R. J. J. Nucl. Med. 2010, 51, 1167.-   Zhou, et al., J. Am. Chem. Soc., 134:7803-7811, 2012.-   Zhou, K. et. al., Angew. Chem. Int. Ed. 2011, 50, 6109-6114-   Zhou, K.; Lu, Y.; Li, J.; Shen, L.; Zhang, G.; Xie, Z.; Wu, C.    Macromolecules 2008, 41, 8927-   Zoncu et al., Science, 334:678-683, 2011.

What is claimed is:
 1. A polymer of the formula:

wherein: R₁ is hydrogen, alkyl_((C≤12)), cycloalkyl_((C≤12)),substituted alkyl_((C≤12)), substituted cycloalkyl_((C≤12)), or

 or a metal chelating group; n is an integer from 1 to 500; R₂ and R₂′are each independently selected from hydrogen, alkyl_((C≤12)),cycloalkyl_((C≤12)), substituted alkyl_((C≤12)), or substitutedcycloalkyl_((C≤12)); R₃ is a group of the formula:

wherein: n_(x) is 1-10; λ₁, λ₂, and λ₃ are each independently selectedfrom hydrogen, alkyl_((C≤12)), cycloalkyl_((C≤12)), substitutedalkyl_((C≤12)), or substituted cycloalkyl_((C≤12)); and λ₄ and λ₅ areeach independently selected from alkyl_((C≤12)), cycloalkyl_((C≤12)),aryl_((C≤12)), heteroaryl_((C≤12)) or a substituted version of any ofthese groups, or λ₄ and λ₅ are taken together and arealkanediyl_((C≤12)), alkoxydiyl_((C≤12)), alkylaminodiyl_((C≤12)), or asubstituted version of any of these groups; x is an integer from 1 to150; R₄ is a group of the formula:

wherein: n_(y) is 1-10; λ₁′, λ₂′, and λ₃′ are each independentlyselected from hydrogen, alkyl_((C≤12)), cycloalkyl_((C≤12)), substitutedalkyl_((C≤12)), or substituted cycloalkyl_((C≤12)); and λ₄′ and λ₅′ areeach independently selected from alkyl_((C≤12)), cycloalkyl_((C≤12)),aryl_((C≤12)), heteroaryl_((C≤12)) or a substituted version of any ofthese groups, or λ₄′ and λ₅′ are taken together and arealkanediyl_((C≤12)), alkoxydiyl_((C≤12)), alkylaminodiyl_((C≤12)), or asubstituted version of any of these groups; y is an integer from 1 to150; R₅ is a group of the formula:

wherein: n_(z) is 1-10; Y₁, Y₂, and Y₃ are each independently selectedfrom hydrogen, alkyl_((C≤12)), cycloalkyl_((C≤12)), substitutedalkyl_((C≤12)), or substituted cycloalkyl_((C≤12)); and Y₄ is hydrogen,alkyl_((C≤12)), acyl_((C≤12)), substituted alkyl_((C≤12)), substitutedacyl_((C≤12)), a dye, or a fluorescence quencher; z is an integer from0-6; and R₆ is hydrogen, halo, hydroxy, alkyl_((C≤12)), or substitutedalkyl_((C≤12)), wherein R₃, R₄, and R₅ can occur in any order within thepolymer, provided that R₃ and R₄ are not the same group.
 2. The polymerof claim 1, further defined by the formula wherein: R₁ is hydrogen,alkyl_((C≤12)), substituted alkyl_((C≤12)), or

or a metal chelating group; n is an integer from 10 to 500; R₂ and R₂′are each independently selected from hydrogen, alkyl_((C≤12)), orsubstituted alkyl_((C≤12)); R₃ is a group of the formula:

wherein: λ₁, λ₂, and λ₃ are each independently selected from hydrogen,alkyl_((C≤12)), or substituted alkyl_((C≤12)); and λ₄ and λ₅ are eachindependently selected from alkyl_((C≤12)), aryl_((C≤12)),heteroaryl_((C≤12)) or a substituted version of any of these groups, orλ₄ and λ₅ are taken together and are alkanediyl_((C≤8)),alkoxydiyl_((C≤8)), alkylaminodiyl_((C≤8)), or a substituted version ofany of these groups; x is an integer from 1 to 100; R₄ is a group of theformula:

wherein: λ₁′, λ₂′, and λ₃′ are each independently selected fromhydrogen, alkyl_((C≤12)), or substituted alkyl_((C≤12)); and λ₄′ and λ₅′are each independently selected from alkyl_((C≤12)), aryl_((C≤12)),heteroaryl_((C≤12)) or a substituted version of any of these groups, orλ₄′ and λ₅′ are taken together and are alkanediyl_((C≤8)),alkoxydiyl_((C≤8)), alkylaminodiyl_((C≤8)), or a substituted version ofany of these groups; y is an integer from 1 to 100; R₅ is a group of theformula:

wherein: Y₁, Y₂, and Y₃ are each independently selected from hydrogen,alkyl_((C≤12)), substituted alkyl_((C≤12)); and Y₄ is hydrogen,acyl_((C≤12)), substituted acyl_((C≤12)), a dye, or a fluorescencequencher; z is an integer from 0-6; and R₆ is hydrogen, halo,alkyl_((C≤12)), or substituted alkyl_((C≤12)), wherein R₃, R₄, and R₅can occur in any order within the polymer, provided that R₃ and R₄ arenot the same group.
 3. The polymer of either claim 1 or claim 2, furtherdefined by the formula wherein: R₁ is hydrogen, alkyl_((C≤8)),substituted alkyl_((C≤8)), or

or a metal chelating group; n is an integer from 10 to 200; R₂ and R₂′are each independently selected from hydrogen, alkyl_((C≤8)), orsubstituted alkyl_((C≤8)); R₃ is a group of the formula:

wherein: λ₁, λ₂, and λ₃ are each independently selected from hydrogen,alkyl_((C≤8)), or substituted alkyl_((C≤8)); and λ₄ and λ₅ are eachindependently selected from alkyl_((C≤12)), aryl_((C≤12)),heteroaryl_((C≤12)) or a substituted version of any of these groups, orλ₄ and λ₅ are taken together and are alkanediyl_((C≤8)) or substitutedalkanediyl_((C≤8)); x is an integer from 1 to 100; R₄ is a group of theformula:

wherein: λ₁′, λ₂′, and λ₃′ are each independently selected fromhydrogen, alkyl_((C≤8)), or substituted alkyl_((C≤8)); and λ₄′ and λ₅′are each independently selected from alkyl_((C≤12)), aryl_((C≤12)),heteroaryl_((C≤12)) or a substituted version of any of these groups, orλ₄′ and λ₅′ are taken together and are alkanediyl_((C≤8)) or substitutedalkanediyl_((C≤8)); y is an integer from 1 to 100; R₅ is a group of theformula:

wherein: Y₁, Y₂, and Y₃ are each independently selected from hydrogen,alkyl_((C≤8)), substituted alkyl_((C≤8)); and Y₄ is hydrogen, a dye, ora fluorescence quencher; z is an integer from 0-6; and R₆ is hydrogen,halo, alkyl_((C≤6)), or substituted alkyl_((C≤6)), wherein R₃, R₄, andR₅ can occur in any order within the polymer, provided that R₃ and R₄are not the same group.
 4. The polymer according to any one of claims1-3, wherein R₁ is hydrogen.
 5. The polymer according to any one ofclaims 1-3, wherein R₁ is alkyl_((C≤6)).
 6. The polymer of claim 5,wherein R₁ is methyl.
 7. The polymer according to any one of claims 1-3,wherein R₁ is


8. The polymer according to any one of claims 1-3, wherein R₁ is a metalchelating group.
 9. The polymer according to any one of claims 1-3,wherein R₁ is a metal chelating group selected from DOTA, TETA, Diamsar,NOTA, NETA, TACN-TM, DTPA, TRAP, NOPO, AAZTA, DATA, HBED, SHBED, BPCA,CP256, DFO, PCTA, HEHA, PEPA, or a derivative thereof.
 10. The polymerof claim 8, wherein the metal chelating group is a nitrogen containingmacrocycle.
 11. The polymer of claim 8, wherein the nitrogen containingmacrocycle is a compound of the formula:

wherein: R₇, R₈, R₉, R₁₀, R₇′, R₈′, and R₉′ are each independentlyselected from hydrogen, alkyl_((C≤12)), acyl_((C≤12)),-alkanediyl_((C≤12))-acyl_((C≤12)), or a substituted version of any ofthese groups; or a linker, wherein the linker is analkanediyl_((C≤12))-C(O)NH— or a substitutedalkanediyl_((C≤12))-C(O)NH—; or R₇ is taken together with one of R₈, R₉,or R₁₀ and is alkanediyl_((C≤6)); or R₈ is taken together with one ofR₇, R₉, or R₁₀ and is alkanediyl_((C≤6)); or R₉ is taken together withone of R₇, R₈, or R₁₀ and is alkanediyl_((C≤6)); or R₁₀ is takentogether with one of R₇, R₈, or R₉ and is alkanediyl_((C≤6)); or R₇′ istaken together with one of R₈′ or R₉′ and is alkanediyl_((C≤6)); or R₈′is taken together with one of R₇′ or R₉′ and is alkanediyl_((C≤6)); orR₉′ is taken together with one of R₇′ or R₈′ and is alkanediyl_((C≤6));and a, b, c, d, a′, b′, and c′ are each independently selected from 1,2, 3, or
 4. 12. The polymer of claim 11, wherein a, b, c, d, a′, b′, andc′ are each independently selected from 2 or
 3. 13. The polymeraccording to any one of claims 8-12, wherein the metal chelating groupis:


14. The polymer according to any one of claims 1-3 and 8-13, wherein themetal chelating complex is bound to a metal ion.
 15. The polymer ofclaim 14, wherein the metal ion is a radionuclide or radiometal.
 16. Thepolymer of claim 14, wherein the metal ion is suitable for PET or SPECTimaging.
 17. The polymer of claim 14, wherein the metal chelatingcomplex is bound to a transition metal ion.
 18. The polymer of claim 14,wherein the metal ion is a copper ion, a gallium ion, a scandium ion, anindium ion, a lutetium ion, a ytterbium ion, a zirconium ion, a bismuthion, a lead ion, a actinium ion, or a technetium ion.
 19. The polymer ofclaim 18, wherein the metal ion is an isotope selected from ^(99m)Tc,⁶⁰Cu, ⁶¹Cu, ⁶²Cu, ⁶⁴Cu, ⁸⁶Y, ⁹⁰y, ⁸⁹Zr, ⁴⁴Sc, ⁴⁷Sc, ⁶⁶Ga, ⁶⁷Ga, ⁶⁸Ga,¹¹¹In, ¹⁷⁷Lu, ²²⁵Ac, ²¹²Pb, ²¹²Bi, ²¹³Bi, ¹¹¹In, ^(114m)In, ¹¹⁴In,¹⁸⁶Re, or ¹⁸⁸Re.
 20. The polymer of claim 17, wherein the transitionmetal is a copper(II) ion.
 21. The polymer of claim 20, wherein thecopper(II) ion is a ⁶⁴Cu²⁺ ion.
 22. The polymer of claim 21, wherein themetal chelating complex is:


23. The polymer according to any one of claims 1-7, wherein R₂ isalkyl_((C≤6)).
 24. The polymer of claim 23, wherein R₂ is methyl. 25.The polymer according to any one of claims 1-24, wherein R₂′ isalkyl_((C≤6)).
 26. The polymer of claim 25, wherein R₂′ is methyl. 27.The polymer according to any one of claims 1-26, wherein R₃ is furtherdefined by the formula:

wherein: λ₁ is selected from hydrogen, alkyl_((C≤8)), or substitutedalkyl_((C≤8)); and λ₄ and λ₅ are each independently selected fromalkyl_((C≤12)), aryl_((C≤12)), heteroaryl_((C≤12)) or a substitutedversion of any of these groups, or λ₄ and λ₅ are taken together and arealkanediyl_((C≤8)) or substituted alkanediyl_((C≤8));
 28. The polymer ofclaim 27, wherein λ₁ is alkyl_((C≤6)).
 29. The polymer of claim 28,wherein λ₁ is methyl.
 30. The polymer according to any one of claims27-29, wherein λ₄ is alkyl_((C≤8)).
 31. The polymer of claim 30, whereinλ₄ is methyl, ethyl, propyl, butyl, or pentyl.
 32. The polymer accordingto any one of claims 27-31, wherein λ₅ is alkyl_((C≤8)).
 33. The polymerof claim 32, wherein λ₅ is methyl, ethyl, propyl, butyl, or pentyl. 34.The polymer according to any one of claims 1-33, wherein R₄ is furtherdefined by the formula:

wherein: λ₁′ is selected from hydrogen, alkyl_((C≤8)), or substitutedalkyl_((C≤8)); and λ₄′ and λ₅′ are each independently selected fromalkyl_((C≤12)), aryl_((C≤12)), heteroaryl_((C≤12)) or a substitutedversion of any of these groups, or λ₄′ and λ₅′ are taken together andare alkanediyl_((C≤8)) or substituted alkanediyl_((C≤8)).
 35. Thepolymer of claim 34, wherein λ₁′ is alkyl_((C≤6)).
 36. The polymer ofclaim 35, wherein λ₁ is methyl.
 37. The polymer according to any one ofclaims 34-36, wherein λ₄′ is alkyl_((C≤8)).
 38. The polymer of claim 37,wherein λ₄′ is methyl, ethyl, propyl, butyl, or pentyl.
 39. The polymeraccording to any one of claims 34-38, wherein λ₅′ is alkyl_((C≤8)). 40.The polymer of claim 39, wherein λ₅′ is methyl, ethyl, propyl, butyl, orpentyl.
 41. The polymer according to any one of claims 1-40, whereineach R₃ is incorporated consecutively to form a block.
 42. The polymeraccording to any one of claims 1-41, wherein each R₄ is incorporatedconsecutively to form a block.
 43. The polymer according to any one ofclaims 1-42, wherein R₃ is present as a block and R₄ is present as ablock.
 44. The polymer according to any one of claims 1-40, wherein R₃and R₄ are randomly incorporated within the polymer.
 45. The polymeraccording to any one of claims 1-44, wherein R₅ is further defined bythe formula:

wherein: Y₁ is selected from hydrogen, alkyl_((C≤8)), substitutedalkyl_((C≤8)); and Y₄ is hydrogen, a dye, or a fluorescence quencher.46. The polymer of claim 45, wherein Y₁ is alkyl_((C≤6)).
 47. Thepolymer of claim 46, wherein Y₁ is methyl.
 48. The polymer according toany one of claims 45-47, wherein Y₄ is hydrogen.
 49. The polymeraccording to any one of claims 45-47, wherein Y₄ is a dye.
 50. Thepolymer of claim 49, wherein Y₄ is fluorescent dye.
 51. The polymer ofclaim 50, wherein the fluorescent dye is a coumarin, fluorescein,rhodamine, xanthene, BODIPY®, Alexa Fluor®, or cyanine dye.
 52. Thepolymer of claim 50, wherein the fluorescent dye is indocyanine green,AMCA-x, Marina Blue, PyMPO, Rhodamine Green™, Tetramethylrhodamine,5-carboxy-X-rhodamine, Bodipy493, Bodipy TMR-x, Bodipy630, Cyanine3.5,Cyanine5, Cyanine5.5, and Cyanine7.5.
 53. The polymer of claim 50,wherein the fluorescent dye is indocyanine green.
 54. The polymeraccording to any one of claims 45-47, wherein Y₄ is a fluorescencequencher.
 55. The polymer of claim 54, wherein the fluorescence quencheris QSY7, QSY21, QSY35, BHQ1, BHQ2, BHQ3, TQ1, TQ2, TQ3, TQ4, TQ5, TQ6,and TQ7.
 56. The polymer according to any one of claims 1-52, wherein nis 75-150.
 57. The polymer of claim 56, wherein n is 100-125.
 58. Thepolymer according to any one of claims 1-57, wherein x is 1-99.
 59. Thepolymer of claim 58, wherein x is from 1-5, 5-10, 10-15, 15-20, 20-25,25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75,75-80, 80-85, 85-90, 90-95, 95-99 or any range derivable therein. 60.The polymer according to any one of claims 1-59, wherein y is 1-99. 61.The polymer of claim 60, wherein y is from 1-5, 5-10, 10-15, 15-20,20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70,70-75, 75-80, 80-85, 85-90, 90-95, 95-99 or any range derivable therein.62. The polymer according to any one of claims 1-61, wherein z is 0-6.63. The polymer according to any one of claims 1-61, wherein z is 1-6.64. The polymer of claim 62, wherein z is from 0-2, 2-4, 4-6, or anyrange derivable therein.
 65. The polymer according to any one of claims1-64, wherein R₃, R₄, and R₅ can occur in any order within the polymer.66. The polymer according to any one of claims 1-64, wherein R₃, R₄, andR₅ occur in the order described in formula I.
 67. The polymer accordingto any one of claims 1-66, wherein the polymer further comprises atargeting moiety.
 68. The polymer of claim 67, wherein the targetingmoiety is a small molecule, an antibody, an antibody fragment, or asignaling peptide.
 69. The polymer according to any one of claims 1-66,wherein R₃ and R₄ are selected from:


70. The polymer according to any one of claims 1-69, wherein the polymeris PEO₁₁₄-P(DEA₂₀-D5A₆₀), PEO₁₁₄-P(DEA₄₀-D5A₄₀), PEO₁₁₄-P(DEA₆₀-D5A₂₀),PEO₁₁₄-P(DPA₆₀-DBA₂₀), PEO₁₁₄-P(DPA₄₀-DBA₄₀), PEO₁₁₄-P(DPA₂₀-DBA₆₀),PEO₁₁₄-P(DEA₇₆-DPA₂₄), PEO₁₁₄-P(DEA₅₈-DPA₄₂), PEO₁₁₄-P(DEA₃₉-DPA₆₁),PEO₁₁₄-P(DEA₂₁-DPA₇₉), PEO₁₁₄-P(DPA₃₀-DBA₅₀), PEO₁₁₄-P(DBA₂₈-D5A₅₂),PEO₁₁₄-P(DBA₅₆-D5A₂₄), PEO₁₁₄-P(DEA₂₀-D5A₆₀-AMA₃),PEO₁₁₄-P(DEA₄₀-D5A₄₀-AMA₃), PEO₁₁₄-P(DEA₆₀-D5A₂₀-AMA₃),PEO₁₁₄-P(DPA₆₀-DBA₂₀-AMA₃), PEO₁₁₄-P(DPA₄₀-DBA₄₀-AMA₃),PEO₁₁₄-P(DPA₂₀-DBA₆₀-AMA₃), PEO₁₁₄-P(DEA₇₆-DPA₂₄-AMA₃),PEO₁₁₄-P(DEA₅₈-DPA₄₂-AMA₃), PEO₁₁₄-P(DEA₃₉-DPA₆₁-AMA₃),PEO₁₁₄-P(DEA₂₁-DPA₇₉-AMA₃), PEO₁₁₄-P(DPA₃₀-DBA₅₀-AMA₃),PEO₁₁₄-P(DBA₂₈-D5A₅₂-AMA₃), PEO₁₁₄-P(DBA₅₆-D5A₂₄-AMA₃),PEO₁₁₄-P(DEA₁₁-EPA₈₉), PEO₁₁₄-P(DEA₂₂-EPA₇₈), PEO₁₁₄-P(EPA₉₀-DPA₁₀), orPEO₁₁₄-P(EPA₇₉-DPA₂₁).
 71. A polymer of the formula:

wherein: R₁ is hydrogen, alkyl_((C≤12)), cycloalkyl_((C≤12)),substituted alkyl_((C≤12)), substituted cycloalkyl_((C≤12)), or

or a metal chelating group; n is an integer from 1 to 500; R₂ and R₂′are each independently selected from hydrogen, alkyl_((C≤12)),cycloalkyl_((C≤12)), substituted alkyl_((C≤12)), or substitutedcycloalkyl_((C≤12)); R₃ is a group of the formula:

wherein: n_(x) is 1-10; λ₁, λ₂, and λ₃ are each independently selectedfrom hydrogen, alkyl_((C≤12)), cycloalkyl_((C≤12)), substitutedalkyl_((C≤12)), or substituted cycloalkyl_((C≤12)); λ₄ is pentyl,n-propyl, or ethyl; and λ₅ is pentyl or n-propyl; x is an integer from 1to 100; R₄ is a group of the formula:

wherein: Y₁, Y₂, and Y₃ are each independently selected from hydrogen,alkyl_((C≤12)), cycloalkyl_((C≤12)), substituted alkyl_((C≤12)), orsubstituted cycloalkyl_((C≤12)); and Y₄ is hydrogen, alkyl_((C≤12)),acyl_((C≤12)), substituted alkyl_((C≤12)), substituted acyl_((C≤12)), adye, or a fluorescence quencher; y is an integer from 1 to 6; and R₅ ishydrogen, halo, hydroxy, alkyl_((C≤12)), or substituted alkyl_((C≤12)).72. The polymer of claim 71, wherein Y₄ is a fluorescent dye.
 73. Thepolymer of claim 72, wherein the fluorescent dye is indocyanine green.74. The polymer of claim 71, wherein the polymer is PEO₁₁₄-P(D5A₈₀),PEO₁₁₄-P(D5A₁₀₀), PEO₁₁₄-P(DPA₈₀), PEO₁₁₄-P(DPA₁₀₀), PEO₁₁₄-P(EPA₈₀),and PEO₁₁₄-P(EPA₁₀₀).
 75. A compound of the formula:


76. A micelle of a polymer according to any one of claims 1-74.
 77. A pHresponsive system comprising a micelle of a first polymer wherein thefirst polymer has a formula according to any one of claims 1-74, whereinz is not 0 and Y₄ is a dye, and wherein the micelle has a pH transitionpoint and an emission spectra.
 78. The pH responsive system of claim 77,wherein the micelle further comprises a second polymer with a formulaaccording to any one of claims 1-74, wherein z is not 0 and Y₄ is afluorescence quencher.
 79. The pH responsive system of claim 78, whereinthe second polymer has the same formula as the first polymer except thatY₄ is a fluorescence quencher.
 80. The pH responsive system of claim 77,wherein the micelle comprises a composition comprising a second polymerwith a formula according to any one of claims 1-74, wherein the secondpolymer has a different formula than the first polymer.
 81. The pHresponsive system of claim 80, wherein Y₄ on the second polymer is adifferent dye than the Y₄ on the first polymer.
 82. The pH responsivesystem of claim 80, wherein the micelle further comprises from 1 to 6additional polymers provided that each polymer is unique that eachpolymer is different than the first polymer and the second polymer. 83.The pH responsive system according to any one of claims 77-82, whereinthe pH transition point is between 3-9.
 84. The pH responsive system ofclaim 83, wherein the pH transition point is between 4-8.
 85. The pHresponsive system of claim 83, wherein the pH transition point isbetween 4-6.
 86. The pH responsive system of claim 83, wherein the pHtransition point is between 6-7.5.
 87. The pH responsive system of claim84, wherein the pH transition point is 4.38, 4.67, 4.96, 5.27, 5.63,5.91, 6.21, 6.45, 6.76, 7.08, or 7.44.
 88. The pH responsive systemaccording to any one of claims 77-87, wherein the emission spectra isbetween 400-850 nm.
 89. The pH responsive system according to any one ofclaims 77-88, wherein the system has a pH response (ΔpH_(0-90%)) of lessthan 1 pH unit.
 90. The pH responsive system of claim 89, wherein the pHresponse is less than 0.25 pH units.
 91. The pH responsive system ofclaim 90, wherein the pH response is less than 0.15 pH units.
 92. The pHresponsive system according to any one of claims 77-91, wherein thefluorescence signal has a fluorescence activation ratio of greater than25.
 93. The pH responsive system of claim 92, wherein the fluorescenceactivation ratio is greater than
 50. 94. A method of imaging the pH of aintracellular or extracellular environment comprising: (a) contacting apH responsive system of claims 77-93 with the environment; and (b)detecting one or more optical signals from the environment, wherein thedetection of the optical signal indicates that the micelle has reachedits pH transition point and disassociated.
 95. The method of claim 94,wherein the optical signal is a fluorescent signal.
 96. The method ofclaim 94 or claim 95, wherein when the intracellular environment isimaged, the cell is contacted with the pH responsive system underconditions suitable to cause uptake of the pH responsive system.
 97. Themethod according to any one of claims 94-96, wherein the intracellularenvironment is part of a cell.
 98. The method of claim 97, wherein thepart of the cell is lysosome or an endosome.
 99. The method according toclaim 94 or claim 96, wherein the extracellular environment is of atumor or vascular cell.
 100. The method according to claim 94 or claim96, wherein the extracellular environment is intravascular orextravascular.
 101. The method of claim 99, wherein the imaging the pHof the tumor environment comprises imaging the sentinel lymph node ornodes.
 102. The method of claim 101, wherein imaging sentinel lymph nodeor nodes allows for the surgical resection of the tumor and staging ofthe tumor metastasis.
 103. The method according to any one of claims99-102, wherein imaging the pH of the tumor environment allowsdetermination of the tumor size and margins.
 104. The method of claim103, wherein imaging the pH of the tumor environment allows for moreprecise removal of the tumor during surgery.
 105. The method of claim103, wherein imaging the pH of the sentential lymph node or nodes allowsfor more precise removal of the sentential lymph node or nodes duringsurgery.
 106. The method of claim 94, wherein the method furthercomprises: (a) contacting the cell with a compound of interest; (b)detecting one or more optical signals in the environment; and (c)determining whether a change in the optical signal occurred followingcontacting the cell with the compound of interest.
 107. The method ofclaim 106, wherein the compound of interest is a drug, antibody,peptide, protein, nucleic acid, or small molecule.
 108. A method ofdelivering a compound of interest to a target cell comprising: (a)encapsulating the compound of interest with a pH responsive system of apolymer of claims 1-74; and (b) contacting the target cell with the pHresponsive system under such conditions that the pH of the target celltriggers the disassociation of the pH responsive system and release ofthe compound, thereby delivering the compound of interest.
 109. Themethod of claim 108, wherein the compound of interest is delivered intothe cell.
 110. The method of claim 108, wherein the compound of interestis delivered to the cell.
 111. The method according to any one of claims108-110, wherein the compound of interest is a drug, antibody, peptide,protein, nucleic acid, or small molecule.
 112. The method according toany one of claims 108-111, comprising administering the pH responsivesystem to a patient.
 113. A method of resecting a tumor in a patientcomprising: (a) administering to the patient an effective dose of a pHresponsive system according to any one of claims 77-93; (b) detectingone or more optical signals for the patient; wherein the optical signalsindicate the presence of a tumor; and (c) resecting the tumor viasurgery.
 114. The method of claim 113, wherein the optical signalsindicate the margins of the tumor.
 115. The method of either claim 113or claim 114, wherein the tumor is 90% resected.
 116. The method ofclaim 115, wherein the tumor is 95% resected.
 117. The method of claim116, wherein the tumor is 99% resected.
 118. The method according to anyone of claims 113-117, wherein the tumor is a solid tumor.
 119. Themethod of claim 118, wherein the solid tumor is from a cancer.
 120. Themethod of claim 119, wherein the cancer is a breast cancer or a head andneck cancer.
 121. The method of claim 120, wherein the cancer is headand neck squamous cell carcinoma.
 122. The method according to any oneof claims 113-122, wherein the pH responsive system is comprised of apolymer of the formula:

wherein: x is an integer from 30 to 150, y is an integer from 1 or 2; xand y are randomly distributed throughout the polymer; and ICG is thefluorescent dye indocyanine green.
 123. A method of treating a cancersusceptible to endosomal/lysosomal pH arrest in a patient comprisingadministering to the patient in need thereof a pH responsive systemaccording to any one of claims 77-93.
 124. The method of claim 123,wherein the cancer is a lung cancer.
 125. The method of claim 124,wherein the cancer is a non-small cell lung cancer.
 126. The methodaccording to any one of claims 123-125, wherein the cancer comprises amutation in the KRAS gene.
 127. The method according to any one ofclaims 123-125, wherein the cancer comprises a mutation in the LKB1gene.
 128. The method according to any one of claims 123-127, whereinthe cancer comprises a mutation in both the KRAS and LKB1 gene.
 129. Themethod according to any one of claims 123-128, wherein the method issufficient to induce apoptosis.
 130. A method of identifying thepresence of a genetic mutation in a cell: (a) contacting a pH responsivesystem comprising two or more micelles of claims 77-93 with the cell orcellular environment; and (b) detecting two or more optical signals fromthe environment, wherein the detection of the optical signal indicatesthat one of the micelles has reached its pH transition point anddisassociated; and (c) correlate the two or more optical signals todetermine the presence of the genetic mutation in the cell.
 131. Themethod of claim 130, wherein the genetic mutation is a mutation in theKRAS gene.
 132. The method of either claim 130 or claim 131, wherein thetwo or more micelles comprises three micelles with a pH transition pointat 6.9, 6.2, and 5.3.
 133. The method according to any one of claims130-132, wherein each of the three micelles is prepared from a polymerselected from PDPA-TMR, PEPA-BDY493, and PDBA-Cy5.
 134. The methodaccording to any one of claims 130-133, wherein the method is performedin vivo and contact a cell comprising administering the one or moremicelles to a patient.
 135. A method of identifying the tumor acidosispathway comprising: (a) contacting a pH responsive system comprising oneor more micelles of claims 77-93 with a cell or a cellular environment;(b) contacting the cell with an inhibitor of the pH regulatory pathway;(c) detecting two or more optical signals from the cell or cellularenvironment, wherein the detection of the optical signal indicates thatone of the micelles has reached its pH transition point anddisassociated; and (d) correlating the two or more optical signals witha modification in the tumor acidosis pathway.
 136. The method of claim135, wherein the inhibitor of the pH regulatory pathway is an inhibitorof a monocarboxylate transporter, a carbonic anhydrase, an anionexchanger, a Na⁺-bicarbonate exchanger, a Na⁺/H⁺ exchanger, or aV-ATPase.
 137. The method of either claim 135 or 136, wherein the one ormore micelles comprise a polymer with two or more fluorophores attachedto the polymer backbone.
 138. The method according to any one of claims135-137, wherein the method comprises one micelle and the micellecomprises two or more polymers with different fluorophores or differentR₃ groups.
 139. The method of claim 138, wherein the micelle comprisestwo or more polymers with different fluorophores and different R₃groups.
 140. A method of imaging a patient to determine the presence ofa tumor comprising: (a) contacting a pH responsive system comprising oneor more micelles of claims 77-93 with the tumor, wherein the micellefurther comprises a metal chelating group at R₁; (b) collecting one ormore PET or SPECT imaging scans; and (c) collecting one or more opticalimaging scans, wherein the detection of the optical signal indicatesthat one of the micelles has reached its pH transition point anddisassociated; wherein the one or more PET or SPECT imaging scans andthe one or more optical imaging scans result in the identification of atumor.
 141. The method of claim 140, wherein the optical imaging scansare collected before the PET or SPECT imaging scans.
 142. The method ofclaim 140, wherein the optical imaging scans are collected after the PETor SPECT imaging scans.
 143. The method of claim 140, wherein theoptical imaging scans are collected simultaneously with the PET or SPECTimaging scans.
 144. The method according to any one of claims 140-143,wherein the imaging scans are PET imaging scans.
 145. The methodaccording to any one of claims 140-143, wherein the imaging scans areSPECT imaging scans.
 146. The method according to any one of claims140-145, wherein the metal chelating group is bound to a ⁶⁴Cu ion. 147.The method of claim 146, wherein the metal chelating group is a nitrogencontaining macrocycle.
 148. The method of claim 147, wherein thenitrogen containing macrocycle is:

wherein: R₇, R₈, R₉, R₁₀, R₇′, R₈′, R₉′ a, b, c, d, a′, b′, and c′ areas defined above.
 149. The method of claim 148, wherein the nitrogencontaining macrocycle is:


150. A polymer of the formula:

wherein: R₁ is a metal chelating group; n is an integer from 1 to 500;R₂ and R₂′ are each independently selected from hydrogen,alkyl_((C≤12)), cycloalkyl_((C≤12)), substituted alkyl_((C≤12)), orsubstituted cycloalkyl_((C≤12)); R₃ is a group of the formula:

wherein: n_(x) is 1-10; λ₁, λ₂, and λ₃ are each independently selectedfrom hydrogen, alkyl_((C≤12)), cycloalkyl_((C≤12)), substitutedalkyl_((C≤12)), or substituted cycloalkyl_((C≤12)); and λ₄ and λ₅ areeach independently selected from alkyl_((C≤12)), cycloalkyl_((C≤12)),aryl_((C≤12)), heteroaryl_((C≤12)) or a substituted version of any ofthese groups, or λ₄ and λ₅ are taken together and arealkanediyl_((C≤12)), alkoxydiyl_((C≤12)), alkylaminodiyl_((C≤12)), or asubstituted version of any of these groups; x is an integer from 1 to150; R₄ is a group of the formula:

wherein: n_(z) is 1-10; Y₁, Y₂, and Y₃ are each independently selectedfrom hydrogen, alkyl_((C≤12)), cycloalkyl_((C≤12)), substitutedalkyl_((C≤12)), or substituted cycloalkyl_((C≤12)); and Y₄ is hydrogen,alkyl_((C≤12)), acyl_((C≤12)), substituted alkyl_((C≤12)), substitutedacyl_((C≤12)), a dye, or a fluorescence quencher; y is an integer from0-6; and R₄ is hydrogen, halo, hydroxy, alkyl_((C≤12)), or substitutedalkyl_((C≤12)), wherein R₃ and R₄ can occur in any order within thepolymer, provided that R₃ and R₄ are not the same group.
 151. Thepolymer of claim 150, wherein R₃ is:

wherein: λ₁, λ₄, and λ₅ are as defined above.
 152. The polymer of eitherclaim 150 or claim 151, wherein λ₁ is alkyl_((C≤12)).
 153. The polymerof claim 152, wherein λ₁ is methyl.
 154. The polymer according to anyone of claims 150-153, wherein λ₄ and λ₅ are taken together and arealkanediyl_((C≤12)) or substituted alkanediyl_((C≤12)).
 155. The polymerof claim 154, wherein λ₄ and λ₅ are taken together and are—CH₂CH₂CH₂CH₂CH₂CH₂-.
 156. The polymer according to any one of claims150-155, wherein R₄ is:

wherein: Y₁ and Y₄ are as defined above.
 157. The polymer of claim 156,wherein Y₄ is a dye.
 158. The polymer of claim 157, wherein Y₄ is afluorescent dye.
 159. The polymer according to any one of claims156-158, wherein Y₁ is alkyl_((C≤12)).
 160. The polymer of claim 159,wherein Y₁ is methyl.
 161. The polymer according to any one of claims150-160, wherein x is 40, 60, 80, 100, or
 120. 162. The polymeraccording to any one of claims 150-161, wherein y is 1, 2, or
 3. 163.The polymer of claim 162, wherein y is
 3. 164. The polymer according toany one of claims 150-163, wherein the polymer isPEO₁₁₄-P(C7A₄₀-r-ICG₃), PEO₁₁₄-P(C7A₆₀-r-ICG₃), PEO₁₁₄-P(C7A₈₀-r-ICG₃),PEO₁₁₄-P(C7A₁₀₀-r-ICG₃), or PEO₁₁₄-P(C7A₁₂₀-r-ICG₃), wherein the PEOgroup is capped with a metal chelating group.